4th International Wroclaw Scientific Meetingsbc.wydawnictwo-tygiel.pl/public/assets/468/4th... ·...

4

th International Wroclaw

Scientific Meetings 09-10 October 2020

4



Editors

Julita Kulbacka

Nina Rembiałkowska

Joanna Weżgowiec

Wroclaw 2020

4th

International Wroclaw Scientific Meetings Wroclaw 09-10 October 2020

Editors

Julita Kulbacka

Nina Rembiałkowska

Joanna Weżgowiec

Typesetting

Monika Maciąg

Kamil Maciąg

Cover design

Marcin Szklarczyk

Nina Rembiałkowska

copy Copyright by Wydawnictwo Naukowe TYGIEL Sp z oo

ISBN 978-83-66489-37-0

Publisher

Wydawnictwo Naukowe Tygiel Sp z oo

ul Głowackiego 35341 20-060 Lublin

wwwwydawnictwo-tygielpl

Scientific Committe

dr inż Dagmara Baczyńska Department of Molecular and Cellular Biology Wroclaw Medical University Poland

dr hab inż Urszula Bazylińska Department of Physical and Quantum Chemistry WUST Poland

dr hab Iwona Bil-Lula Department of Medical Laboratory Diagnostics Wroclaw Medical University Poland

dr hab inż Anna Choromańska Department of Molecular and Cellular Biology Wroclaw Medical University Poland

dr Agnieszka Chwiłkowska Department of Molecular and Cellular Biology Wroclaw Medical University Poland

prof dr hab inż Marcin Drąg Department of Bioorganic Chemistry WUST Poland

dr Małgorzata Drąg-Zalesińska Department of Human Morphology and Embryology Wroclaw Medical University

Poland

dr hab Izabela Fecka Department of Pharmacognosy and Herbal Medicines Wroclaw Medical University

Poland

prof dr hab Halina Grajeta Department of Food Science and Dietetics Wroclaw Medical University Poland

dr hab Bożena Karolewicz Department of Drugs Form Technology Wroclaw Medical University Poland

dr hab inż Marta Kepinska prof Department of Biomedical and Environmental Analyses Wroclaw Medical University

Poland

dr Tabassum Khan Nanavati College of Pharmacy India

prof dr hab Reneacute Kizek UVPSB Czechia

prof dr hab inż Małgorzata Kotulska Department of Biomedical Engineering WUST Poland

dr hab inż Julita Kulbacka prof nadzw Department of Molecular and Cellular Biology Wroclaw Medical University Poland

Marek Kulbacki DIVE IN AI Polish Japanese Academy of Information Technology Poland

dr Łukasz Lamch Department of Engineering and Technology of Chemical Processes WUST Poland

prof dr hab Adam Matkowski Department of Biology and Pharmaceutical Botany Wroclaw Medical University Poland

prof dr hab Halina Milnerowicz Department of Biomedical and Environmental Analyses Wroclaw Medical University Poland

dr Helena Moreira Department of Basic Medical Sciences Wroclaw Medical University Poland

prof dr Vitalij Novickij Faculty of Electronics Vilnius Gediminas Technical University Lithuania

prof Olga Pakhomova Old Dominion University USA

prof dr hab Agnieszka Piwowar Department of Toxicology Wroclaw Medical University Poland

dr inż Nina Rembiałkowska Department of Molecular and Cellular Biology Wroclaw Medical University Poland

dr inż Joanna Rossowska Institute of Immunology and Experimental Therapy Poland

prof dr hab Jolanta Saczko Department of Molecular and Cellular Biology Wroclaw Medical University Poland

prof Mounir Tarek CNRS ndash University de Lorraine Nancy France

dr Joanna Tunikowska Wrocław University of Environmental and Life Sciences Poland

Organizing Committe


mgr Katarzyna Bieżuńska-Kusiak Department of Molecular and Cellular Biology Wroclaw Medical University Poland

dr hab Anna Bizon Department of Biomedical and Environmental Analyses Wroclaw Medical University

Poland



mgr Agata Goacuterska Department of Molecular and Cellular Biology Wroclaw Medical University Poland


Poland

lek Aleksander Kiełbik Department of Molecular and Cellular Biology Wroclaw Medical University Poland


dr hab Ewa M Kratz Department of Laboratory DiagnosticsWroclaw Medical University Poland

dr Adriana Kubis-Kubiak Department of Toxicology Wroclaw Medical University Poland



dr hab inż Sebastian Kraszewski Department of Biomedical Engineering WUST Poland

prof dr hab Adam Matkowski Department of Biology and Pharmaceutical Botany Wroclaw Medical University

Poland

dr Olga Michel Department of Molecular and Cellular Biology Wroclaw Medical University Poland



Edyta Podolan Department of Molecular and Cellular Biology Wroclaw Medical University Poland

lek Dawid Przystupski Department of Molecular and Cellular Biology Wroclaw Medical University Poland

dr inż Łukasz Radosiński Department of Chemistry WUST Poland



dr Ewa Sawicka Department of Toxicology Wroclaw Medical University Poland

dr Anna Szewczyk Department of Molecular and Cellular Biology Wroclaw Medical University Poland

Wojciech Szlasa Department of Molecular and Cellular Biology Wroclaw Medical University Poland

dr Beata Szymańska Department of Toxicology Wroclaw Medical University Poland

Karolina Tądel Wroclaw Medical University Poland

dr inż Joanna Weżgowiec Department of Experimental Dentistry Wroclaw Medical University Poland

lek Piotr Wawryka Department of Molecular and Cellular Biology Wroclaw Medical University Poland

Organizers

Honorary Patronage

Rector of Wroclaw Medical University

Prof dr hab Piotr Ponikowski

Vice-Rector for Educational Affairs

Prof dr hab Agnieszka Piwowar

Sponsors

SHIM-POL AM BORZYMOWSKI Ul Lubomirskiego 5

05-080 Izabelin

NIP 118-178-86-00

tel22722-70-48 do 50

fax 22722-70-51

wwwshim-polpl

Shim-Pol AM Borzymowski provides a wide spectrum of devices and accessories

in the area of chromatography spectroscopy optics mass spectrometry bioanalyzers advanced

surface analyzers as well as testing machines We organize numerous educational meetings

and trainings the Academy of Analytical Chemistry Shim-Pol Day meetings workshops

with LabSolutions software chromatography courses and apparatus demonstrations

Santander Bank Polska Foundation

Corporate volunteering grant programmes the Flicker Club and the

Scholarship Programme are the key initiatives pursued by the IJ

Paderewski Santander Bank Polska Foundation

For over 20 years the Santander Bank Polska Foundation has been helping children

teenagers senior citizens and people in need as well as driving through green changes all

over Poland In 2010 the Foundation started its corporate volunteering project in order to

financially support the initiatives of its bank employees Today around 2000 volunteers are

working with the Foundation to aid the most destitute people

The Santander Bank Polska Foundation is also running two grant programmes February

2020 saw the beginning of a new grant competitionECO-FRIENDLY Here I live here

I make change which helps local communities make their neighbourhood more

environment-friendly The other grant competition Bank of Young Sports Champions

promotes sports activities and a healthy lifestyle among children and teenagers In addition

the Foundation is working on the Flicker Club project This initiative aims to turn unused

rooms in hospitals and foster homes into new beautiful and colourful space for children and

their parents The continued support provided by the Foundation to gifted young people

inspired the launch of the Scholarship Programme For 2 years the Foundation has been

receiving applications from students from all over Poland selecting the most talented ones

and helping them financially to develop their potential In 2004 the Foundation was granted

the status of a non-profit organisation Its activities are supported by a number of taxpayers

who decide to donate 1 of their taxes to the Foundation Those funds enable the

Foundation to help needy people and launch more and more new projects whose effects are

visible throughout Poland

Sygnis Bio Technologies sp z oo wwwsygnisplbio

biosygnispl

+48 22 668 47 57

Żwirki i Wigury 101 Str

02-089 Warsaw Poland

In Sygnis Bio Technologies we strive to provide our partners with the most inventive

approach to bioengineering We are a leading provider of scientific equipment on the Polish

market with a strong focus on high resolution live cell imaging and tissue engineering Our

portfolio combines a multitude of techniques ie 3D bioprinting biopatterning live cell and

super-resolution imaging

Content

CONFERENCE PROGRAM 17

LECTURES 23

ORAL PRESENTATIONS 31

POSTER PRESENTATIONS 75

INDEX OF AUTHORS 276

CONFERENCE PROGRAM

19

09102020 Friday

0900-0910 Opening ceremony ndash Prof Jolanta Saczko and Prof Agnieszka Piwowar (Vice-Rector for Educational Affairs)

1st SESSION

Moderators Jolanta Saczko and Agnieszka Piwowar 0910-0955 Lecture of Prof Mounir Tarek (CNRS ndash University de Lorraine Nancy France) Frontiers of Electroporation from Mechanisms to Applications Unraveling new key molecular level aspects using computational chemistry 1000-1100 Presentations of young scientists S1O1 Relationship between the concentration of IL-6 insulin activity glycated haemoglobin in human blood and the development of type 2 diabetes andor obesity Magdalena Kroacutel S1O2 Assessment of the stimulation level of the antitumor response by dendritic cells modified to overexpression of IL-12 andor IL-18 in vitro Węgierek Katarzyna S1O3 Development of new polymeric materials with the incorporated API for potential application in solid dosage forms formulation using 3D printing technology Kozakiewicz Marta S1O4 Antitumor activity of therapy composed of methotrexate nanoconjugate and dendritic cell-based vaccines and its influence on local and systemic antitumor immune response Szczygieł Agnieszka S1O5 Regulation of mitochondrial dynamics and mitophagy in 2-methoxyestradiol-mediated osteosarcoma ndash and glial-cell death Przychodzen Paulina S1O6 Alterations in plasma concentration and activity of superoxide dismutases in context of obesity andor type 2 diabetes Lewandowski Łukasz

2nd

SESSION Moderators Rene Kizek and Helena Moreira

1115-1145 Lecture of Prof Rene Kizek (University of Brno Czech Republic) Nanomedicine for targeted treatment of tumor diseases 1150-1250 Presentations of young scientists S2O1 Effect of the surface charge on the structure of lysosome adsorbed on the gold surface Komorek Paulina S2O2 Enhancement of antimicrobial photodynamic therapy using metallic nanoparticles Wanarska Ewelina S2O3 Bile salts loaded Lipid Liquid Crystalline Nanoparticles for topical administration of natural antioxidants Fornasier Marco S2O4 Functionalization of curdlan gel for effective antibiotic bonding Michalicha Anna S2O5 Cytostatic effects of natural products on rhabdomyosarcoma ndash in vitro studies Kazimierczak Weronika 1300-1345 Flow Cytometry Webinar ndash Workshop (Sysmex Joanna Rossowska and Helena Moreira)

20

3rd

SESSION Moderators Joanna Tunikowska and Joanna Rossowska

1350-1420 Lecture of Dr Joanna Tunikowska (Wrocław University of Environmental

and Life Sciences Poland) Electrochemotherapy and lasers in surgical battle with tumors

in dogs and cats

1425-1525 Presentations of young scientists

S3O1 Determination of genetically modified dendritic cellsrsquo ability to survive migrate

to the lymph nodes and infiltrate the tumor tissue of MC38 colon carcinoma in in vitro

and in vivo study Mierzejewska Jagoda

S3O2 Knowledge about coronary artery disease among Polish students ndash survey study

Kanclerz Gabriela

S3O3 Development and evaluation of microsponge based topical gel for acne Sanjana

Menezes

S3O4 Fluorescent polymeric nanocarriers of low cytotoxicity for two-photon

bioimaging Nawrot Katarzyna

S3O5 Mixture of MMP-2 MLCK and NOS inhibitors induce cardioprotection against

myocardial ischemiareperfusion injury Krzywonos-Zawadzka Anna

S3O6 Personalization of pancreatic cancer treatment- electroporation

electrochemotherapy and calcium electroporation pilot study Rudno-Rudzińska Julia

1530-1600 ndash Lecture of Dr Tabassum Khan (Nanavati College of Pharmacy India)

At the crossroad of drug discovery and delivery ndash PARP inhibitors and hollow metallic

nanoparticles in cancer therapeutics

10102020 Saturday

4

th SESSION

Moderators Jolanta Saczko and Julita Kulbacka


S4O1 Thiazole based derivatives as sirtuins inhibitors Molecular docking study

Czyżnikowska Żaneta

S4O2 Fighting cancer with gravity Przystupski Dawid

S4O3 Combination of EP and PDT in melanoma treatment Szlasa Wojciech

S4O4 Pulsed Current Evaluation for Prediction of Tumor Permeabilization Rate

Malyško Veronika

S4O5 A multifunctional nanoprobe based on polymeric nanocapsules loaded with

quantum dots and lanthanide doped nanocrystals Antoniak Magda

S4O6 Light-induced in situ TEM microscopy revealing ultrastructural interactions

in antimicrobial photodynamic therapy Andrzej Żak

21

5th

SESSION

Webinar ndash 3D Bio-printing and holotompgraphic microscopy 1010-1040 Leader Grzegorz Kaszyński (Sygnis BioTechnolgies)

6th

SESSION Moderators Olga Pakhomova and Julita Kulbacka

1045-1115 Lecture of Prof Olga Pakhomova (Old Dominion University USA) Calcium-mediated plasma membrane repair

1120-1300 Presentations of young scientists S6O1 Changes in nuclear proteome associated with lamin in the Drosophila melanogaster model system after heat induction Pałka Marta S6O2 Antiviral activity of extract of Ginkgo biloba (EGb) and its phytochemical constituents against herpesviruses HHV-1 and HHV-2 Sochocka Marta S6O3 Trimethylammonium 1-mercapto-1-carbadodecarnorate (TMA) ndash pharmaceutical precursor in BNCT method Woacutejciuk Karolina S6O4 The structure of Nucleobindin-2 is regulated by divalent metal cations Skorupska Anna S6O5 Stress affects the expression of lsquomajor housekeepingrsquo genes ndash is phosphorylation involved Tomczak Aleksandra S6O6 Male infertility in context of oxidative stress the analysis of Total Antioxidant Status and clusterin concentration in human seminal plasma ndash pilot study Janiszewska Ewa S6O7 Volatile compounds as a means of protecting bacterial contamination of cosmetics Surowiak Alicja K S6O8 Studies on the reactivity of human serum IgG and IgA antibodies with the bacterial OmpC protein as a potential diagnostic marker of humoral immuno-deficiency in children Naporowski Piotr

7th

SESSION ndash Molecular Biology Workshop

Leader Katarzyna Widerak (Syngen Biotech)

1330-1415 ndash MB webinar training

8th

SESSION ndash Molecular Dynamics Workshop

Leader Mounir Tarek and Sebastian Kraszewski 1430-1515 ndash MD webinar training

available all the time VIRTUAL POSTER SESSION

1545 Announcement of the results for the best oral and poster presentations and closing ceremony

LECTURES

25

L 1

Frontiers of Electroporation from Mechanisms to Applications

Unravelling new key molecular level aspects using computational

chemistry

Mounir Tarek

Centre National de La Recherche Scientifique (CNRS) Universiteacute de Lorraine Nancy

FRANCE

The application of short and intense electric pulses enables to transiently alter the properties

of cell membranes making them permeable to a wide range of chemical species This

phenomenon is routinely used in a range of medical applications as well in biotechnology

and industrial processing Few years ago pioneering MD simulations have been conducted

in order to model the effect of electric fields on membranes providing perhaps the first

molecular model of the electroporation process of lipid bilayers Our knowledge however

about all occurring processes is still sketchy In this contribution we show how we harness

the capabilities of computational resources and the predictive power of advanced atomistic

and quantum level molecular dynamics techniques to decipher key steps in several physical

and biophysical and chemical processes occurring at the cell membranes when these are

subject to electric pulses used in Electroporation Based Technologies and Treatments

26

L 2

Nanomedicine for targeted treatmentof tumor diseases

Rene Kizek

Department of Human Pharmacology and Toxicology University of Veterinary and Phar-

maceutical Sciences Brno Palackeacuteho třiacuteda 19461 612 42 Brno Czech Republic

Cancer is the second leading cause of death in developed countries It is known that standard

antitumor therapy has a number of serious adverse biological effects One of these is the lack

of selectivity for tumor tissue resulting in significant side effects The relatively low

therapeutic concentration of the active compound often results in drug resistance and multi-

resistance of tumor cells Nanotransporters for targeted treatment are a modern and effective

way of personalized approach Carbon gold silver and other nanoparticles can be used as

the basis of the nanotransporter Nanoparticles can enter a cell independently of its type and

functional group attached to the surface of the nanoparticle Various in vitro and in vivo

studies have shown that many functionalized nanoparticles are biocompatible The physico-

chemical properties of nanoparticles play a decisive role in their potential toxicity For

carbon nanoparticles shorter and thicker nanotubes have been found to exhibit lower

toxicity Chemically functionalized carbon nanotubes (CNTs) are much better water-soluble

and have greater stability in the physiological environment Attempts to use CNTs to target

multivalent ligands in cancer are increasing rapidly In addition to passive targeting methods

based on the enhanced permeability and retention (EPR) effect and the specific acidic

environment in the tumor strategies for actively targeting a selected tumor using ligands

or antibodies that increase the specificity of the nanotransporter are also investigated

However a protein corona plays a major role in the application of nanoparticles in vivo

A protein corona is a cluster of all proteins that can bind to nanoparticles Protein corona

formation is usually associated with a significant reduction in therapeutic potential Albumin

is the most abundant component of the protein corona It has been shown that the

composition of the protein corona depends on the structure and physico-chemical properties

of the nanoparticles However the effect of surfactants on the structure of CNTs on the

composition and formation of the protein corona has not yet been studied In our

experimentsthe effect of the interaction of bovine serum albumin (BSA) and CNTs was

studied A completely unanswered question is the interaction of nanoparticles with thiol

compounds such as low-molecular-weight glutathione or metallothionein In addition to the

above in some malignant tumors we observe increased expression of albumin receptors

(liver gallbladder but also breast cancer) This may be advantageous for nanoparticles with

a protein corona Research in this area of nanomedicine is completely open and will

certainly bring many unexpected discoveries in the near future

27

L 3

Electrochemotherapy and lasers in surgical battle with tumors in dogs

and cats

Joanna Tunikowska

Department of Surgery Faculty of Veterinary Medicine Wrocław University of Environmental

and Life Sciences Wroclaw Poland

A successful treatment of patients with solid tumors consists of three components complete

tumor removal with tumor-free margins identification and removal of tumor-positive lymph

nodes and removal of any local satellite tumor deposits (Holt et al 2015) Therefore the

vast majority of both human and veterinary oncologic patients require combination therapy

surgery and additional oncologic treatment While the method of tumor excision depends

mainly from surgeon the decision on additional oncologic treatment relies on patient general

health status and tumor characteristics Ten privately owned dogs and four cats presented to

the Department of Surgery Faculty of Veterinary Medicine WUELES with confirmed

malignant tumors were treated both laser surgery and electrochemotherapy Laser CO2 is

one of the most universal surgical lasers allowing for precise excision and ablation of soft

tissue with simultaneous sealing of small blood vessels lymph vessels and nociceptors

(Bartels 2002) In addition to surgical treatment local electrochemotherapy (ECT) was

implemented before during or after surgery ECT is a local tumor treatment modality

facilitating intracellular delivery of non-permeant chemotherapeutic drugs followed by the

delivery of electrical pulses to the tumor The advantages of ECT therapy are its simplicity

short duration of treatment sessions low chemotherapeutic doses and insignificant side

effects with excellent functional and cosmetic effects (Tozon Tamzali amp Cemažar 2017)

The combination of both laser CO2 and ECT therapy can be successfully applied as

a curative or palliative treatment also in animals with poor health status

References [1] Bartels K E (2002) Lasers in veterinary medicine ndash Where have we been and where are we going

Veterinary Clinics of North America -Small Animal Practice 32(3) 495ndash515 httpsdoiorg101016S0195-

5616(02)00002-5

[2] Holt D Parthasarathy A B Okusanya O Keating J Venegas O Deshpande C Singhal S (2015)

Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells

inwounds Journal of Biomedical Optics 20(7) 076002 httpsdoiorg1011171jbo207076002

[3] Tozon N Tamzali Y amp Cemažar M (2017) Electrochemotherapy in veterinary oncology Handbook

of Electroporation 3 1953-1967 httpsdoiorg101007978-3-319-32886-7_107

28

L 4

At the crossroad of drug discovery and delivery-PARP inhibitors

and hollow metallic nanoparticles in cancer therapeutics

Tabassum Asif Khan

Department of Pharmaceutical Chemistry amp QASVKMrsquos DrBhanuben Nanavati College

of Pharmacy Mumbai India

Development of PARP inhibitors as cytotoxic leads in cancer therapeutics Poly ADP-ribose

polymerase-1 (PARP-1) is a nuclear enzyme essential to the repair of single strand DNA

breaks via the base excision repairsingle strand break repair pathway Our lab has

synthesized a series of heterocyclic compounds as potential PARP-1 inhibitors Among

these quinazolinone scaffold based analogues have shown good activity A series of twelve

2-styryl quinazolin-4(3H)-one analogues were synthesized by condensation of 2-methyl

quinazolin-4(3H)-one with appropriate aromatic aldehydesThis scaffold was further used to

synthesize a series of hybrids with pyrimidine analoguesThey were screened for cytotoxicity

against MCF-7 cellsusing sulforhodamine B assay The percentage yieldof synthesized

compounds wasfoundto be in the range of 60 to 90 The GI50 of 4-nitrophenyl ethan-1-

ene quinazolin-4-(3H)-onewas found to be 81μgmL comparable to standard Adriamycin

Flow cytometry study for the 4-nitrophenyl ethan-1-ene quinazolin-4-(3H)-one

and 2-nitrophenyl ethan-1-ene quinazolin-4-(3H)-one indicated that the cells in early

apoptosis were ~20 indicating caspase mediated death Few of these along with the hybrid

series werefound to be potent with good PARP-1 inhibitory activityDesign and synthesis of

functionalized hollow metallic nanoparticlesin cancer therapeuticsMetallic nanostructures

with hollow interiors are excellent agents for biomedical applications due to their Surface

Plasmon Resonance (SPR) in Vis-NIR range We report for the first time an extensive study

on the effect of (1) the addition sequence of stabilizer (2) type of stabilizer

(3) the concentration of reducing agent (NaBH4) and(4) the reaction temperature on the SPR

characteristic of glutathione-capped hollow silver nanoparticles (GSH-HAgNPs) using the

sacrificial Ag2O template The photoablation of functionalized metallic nanoparticles (SPR

at 531nm)using a 532 nm NdYAG 300 mW continuous wave (CW) laser led to a 5-6degC

elevation in temperature above physiological temperaturewithin 15 minutessuggesting the

use of GSHas hyperthermia-inducing agent This study provides an evidence of the potential

application of functionalized hollow metallic nanoparticles inbiomedicine especially as drug

delivery cargoes in cancer therapeutics

29

L 5

Calcium-mediated plasma membrane repair

Olga Pakhomova

Frank Reidy Research Center for Bioelectrics Old Dominion University Norfolk Virginia

USA

The plasma membrane isolates intracellular content from the environment and has the

regulatory mechanisms to keep the cell homeostasis unperturbed The membrane injuries

destroy tight control over the cell metabolism and trigger a range of events varying from

moderate stimulatory action to significant disorders in the homeostasis or even cell death

Membrane damages are a common threat to the life of the cells especially for ones

originating in the tissues exposed to mechanical or shear stress (muscles lung vasculature)

or pore-forming toxins In several medical applications (ultrasound or electroporation)

impairing the plasma membrane barrier is a goal and used for drug delivery or tissue

elimination The cells have healing machinery for efficient repair of the membrane damages

Just a few seconds are needed to reseal a membrane pore or remove a membrane tear

Several different models are proposed for membrane restoration All of them consider

the calcium ion as the critical trigger for the repair response activation The lecture aims at

reviewing different cellular and molecular mechanisms of membrane repair with emphasis

on its relevance to disease

ORAL PRESENTATIONS

33

Session 1 O 1

Relationship between the concentration of IL-6 insulin activity

glycated haemoglobin in human blood and the development of type 2

diabetes andor obesity

Magdalena Kroacutel1 Iwona

Urbanowicz

2 Łukasz Lewandowski

3 Marta Kepinska

3

Halina Milnerowicz3

1Students Scientific Association Department of Biomedical Environmental Analyses

Faculty of Pharmacy Wrocław Medical University 2Department of Medical Laboratory

Diagnostics Faculty of Pharmacy Wrocław Medical University 3Department of Biomedical

and Environmental Analyses Faculty of Pharmacy Wrocław Medical University

Background

Nowadays obesity is a very serious problem

in our society Moreover it may be associated

with various metabolic diseases such as

type 2 diabetes (T2D) Many studies indicate

the role of interleukine-6 (IL-6) in the

pathophysiology of T2D Increased level

of IL-6 is an independent predictor of this

disease and is considered to be involved in

the development of inflammation insulin

resistance and β-cell dysfunction On the

other hand recent research suggests that IL-6

has an anti-inflammatory role and improves

glucose metabolism [1 2]

Material and Methods

In this study we analyzed relationship

between the concentration of IL-6 insulin

activity and other selected parameters and

the occurrence of T2D andor obesity These

variables were assayed for human blood

obtained from 117 patients of which 44

respondents belonged to obese without

diabetic group and 23 respondents belonged

to diabetic group For comparison all para-

meters were also measured in the control

group which consisted of 50 respondents

The allocation to the obese group was based

on the BMI value In turn we used the

values of glycated haemoglobin as a marker

of diabetes to assign to the diabetic group

Additional factors considered in this study

were sex and exposition to cigarette smoke

The IL-6 concentration and insulin activity

in serum were tested using suitable ELISA

kits

Results

We observed significantly higher insulin

levels in respondents with obesity compared

to the control group regardless of sex

In addition we noticed differences in IL-6

levels depending on whether the patient was

obese or not However these differences

were not statistically significant

Interestingly we didnrsquot observe statistically

significant higher levels of IL-6 in respon-

dents suffering from T2D

Discussion and conclusions

This research as well as available literature

may show the important role of IL-6 and the

other parameters such as insulin in the

development of obesity and consequently

contribute to the incidence of T2D [3]

Bastard et al hypothesized that adipose tissue

may play a role in the regulation of serum

C-reactive protein concentration through

IL-6 production [4]

Based on the available information relating

to IL-6 its concentration seems to be a good

indicator of activation of the inflammatory

cascade and a predictor of subsequent organ

dysfunction However our data exactly as

in Al-Shukaili et al research didnrsquot show

increased IL-6 levels in serum of patients

34

with diabetes [5] Perhaps this was due

to the small sample size In any case a

better understanding of these mechanisms

can support prevention and treatment of

obesity and T2D

References [1] M Abkari V Hassan-Zadeh et al

Inflammopharmacology vol 26 3 2018 DOI

101007s10787-018-0458-0

[2] K Rehman M S H Akash et al Critical

Reviewstrade in Eukaryotic Gene Expression vol 27

3 2017 DOI 101615CritRevEukaryotGeneExpr

2017019712

[3] K Eder N Baffy et al Agents and Actions vol

58 11 2009 DOI 101007s00011-009-0060-4

[4] J P Bastard C Jardel et al Circulation vol

99 16 1999

[5] A Al-Shukaili S Al-Ghafri et al International

Journal of Endocrinology vol 2013 2013 DOI

1011552013976810

helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip

Session 1 O 2

Assessment of the stimulation level of the antitumor response by dendritic

cells modified to overexpression of IL-12 andor IL-18 in vitro

Katarzyna Węgierek1 Jagoda Mierzejewska

1 Magdalena Geneja

1 Natalia

Anger-Goacutera1 Agnieszka Szczygieł

1 Joanna Rossowska

1 Elżbieta Pajtasz-Piasecka

1

1Ludwik Hirszfeld Institute of Immunology and Experimental Therapy Polish Academy

of Sciences Wroclaw Poland

Background Dendritic cells (DCs) ndash the most effective antigen presenting cells ndash are one of the therapeutic tools to enabling changes in the tumor microenvironment [1 2] The effect of IL-18 on the modulation of the immune response to a large extent depends on the presence of IL-12 in the environment Owing to this fact it seems reasonable to combine these elements in order to elaborate new strategies of cancer-treat-ment DCs gene-tically modified to produce cytokine such as IL-12 and IL-18 may promote the activation of anti-tumor response [3 4] The main objective of these study was to determine the stimulation level of the antitumor response by DCs modified to simultaneous overex-pression of IL-12 and IL-18 stimulated with tumor antigens (TAg) in MC38 colon cancer immunotherapy

Material and Methods Dendritic cells were generated from bone marrow (BM) collected from healthy C57BL6 mice After seven days of culture

BM-DCs were co-transduced with il12 and il18 genes andor treated with MC38 tumor lysate (TAg) On the 9th 11th 13th and 15th days of culture cytokine gene expression (real-time PCR) cytokine production (ELISA) and expression of DCs surface markers (flow cytometry) were evaluated In addition changes in percentage of spleen cell populations (CD4+ CD8+ NK cells CD107a+) from 5-days mixed cultures BM-DC and cytotoxic activity of splenocytes (flow cytometry) were examined

Results The high level of il12 and il18 gene expression and production of these cytokines by transduced BM-DCs were observed Moreover significant changes in the expression of co-stimulatory molecules depending on the type of transduction and length of culture were determined Further-more splenocytes preincubated with BM-DCs transduced for production of cytokines and stimulated with TAg in mixed culture revealed an increase in tumor antigen-specific cytotoxicity In addition an increase

35

in the percentage of CD4+ T cells NK cells and CD107a+ in mixed culture of sple-nocytes and BMDCs transduced for cytokine production were observed

Discussion and conclusions The received results suggest that DCs transduced with il12 and il18 genes additionally stimulated with tumor antigens are able to trigger an antitumor response in vitro and ex vivo This suggests their potential in generation of various DC-based vaccines for anti-tumor immumotherapy

This study was funded by National Science Centre Poland (projects no 201517N NZ402834 201727BNZ602702)

References [1] Steinman R M Banchereau J Nature vol 449419-26 2007 [2] Palucka K Banchereau J Nat Rev Cancer vol 12 265-277 2012 [3] Lasek W et al Cancer Immunol Immunother vol 63 419-435 2014 [4] Srivastava S et al Curr Med Chem vol 17(29) 3353-7 2010


Session 1 O 3

Development of new polymeric materials with the incorporated API

for potential application in solid dosage forms formulation using 3D

printing technology

Marta Kozakiewicz1 Maciej Gajda

1 Adrianna Złocińska

2 Bożena Karolewicz

1

Karol P Nartowski1

1Department of Drug Forms Technology Faculty of Pharmacy Wrocław Medical

University Borowska 211 Wrocław Poland 2Laboratory of Elemental Analysis and

Structural ResearchWrocław Medical University Borowska 211 Wrocław Poland

Background

3D printing is a potential technology suitable

for overcoming limitations for development

and manufacturing of personalized medici-

nes The biggest challenge for wide appli-

cation of 3DP technologies in personalised

medicines is lack of robust lsquoready to usersquo

materials of pharmaceutical quality which

could easily be used in this process [1]

The aim of this work was to synthesize new

polymeric materials with incorporated API

(Active Pharmaceutical Ingredient) which

can be used for FDM 3D printing method

(Fused Deposition Modeling)

In this work we design drugpolymerplas-

ticizer blends which after processing using

hot melt extrusion (HME) formed plastic

filaments ready to use in FDM based 3DP

methods

Material and Methods Amlodipine (AMLO) and hydrochloro-thiazide (HTZ) (Polaura Olsztyn Poland) simvastatin (SIM) and nicotinamide (NIC) (Sigma Aldrich) polymers (HPMCAS 126 AQOATreg type AS-HF and AS-LF Shin-Etsu) and obtained filaments were tested for thermal stability prior HME processing using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) The structural changes of the APIs that might occur during manufacturing process were investigated using X-ray powder diffraction (PXRD) and Fourier Transform Infrared Spectroscopy (FTIR) The filaments were prepared using twin screw hot melt extrusion (Thermo Scientific Process 11 Extruder) and a series of placebo tablets and single-component tablets with simvastatin were printed using the 3D printer Builder Premium

36

Results

Two types of placebo filaments were

produced from HPMCAS AS-HF (I) and

AS-LF (II) polymers and five different

types of filaments with incorporated

therapeutic substances SIM+ HPMCAS

HF(A) SIM + AMLO + HPMCAS HF (B)

AMLO + HTZ + HPMCAS LF (C) AMLO

+ HTZ + Crospovidone + HPMCAS LF

(D) Cocrystal HTZNIC + HPMCAS LF

(E) Two series of tablets using obtained

filament were successfully printed placebo

HPMCAS HF series and single-component

tablets with SIM

The improvement of the plasticity of the

fabricated filaments through the addition of

plasticizer enabled 3D printing process

Modification of melting temperature by

production a co-crystal to adapt thermal

properties to the process window was

presented [2]


Thermal and mechanical properties (ie

thermal stability and glass transition) of the

materials are the key parameters enabling

the use of polymers and APIs in the process

of hot melt extrusion and 3D printing by the

FDM method The plasticity of a filament

with incorporated API depends on the phase

of the drug in the formed filament (ie

crystalline or amorphous) which has been

proven in our work The occurrence of the

API in an amorphous state caused increase

of elasticity of filaments whereas the

presence of drug crystals incorporated in the

filament increased the brittleness and

fragility of the extrudates

The obtained results confirm the possibility of

producing pharmaceutical-quality fila-ments

using hot extrusion and their subsequent use

in the manufacturing of solid oral dosage

forms using FDM 3D printing technology

This research was funded by the Ministry of

Science and Higher Education Poland

through the National Fund for Scientific

Research (Grant No STD 19018001)

References [1] Basit AW Goyanes A Awad A Trenfield SJ

Gaisford S 3D Printing Pharmaceuticals Drug

Development to Frontline Care Trends Pharmacol

Sci 2018 doi101016jtips 201802006

[2] Sanphui P Rajput L Tuning solubility and

stability of hydrochlorothiazide co-crystals Acta

Crystallogr Sect B Struct Sci Cryst Eng Mater

201470(1)81-90doi101107S2052520613026917

Fig1 (A) Filament with incorporated simvastatin (B) Printed tablets with simvastatin


37

Session 1 O 4

Antitumor activity of therapy composed of methotrexate

nanoconjugate and dendritic cell-based vaccines and its influence

on local and systemic antitumor immune response

Agnieszka Szczygieł1 Katarzyna Węgierek

1 Natalia Anger-Goacutera

1 Jagoda

Mierzejewska1 Tomasz Goszczyński

1 Marta Świtalska

1 Joanna Rossowska

1

Elżbieta Pajtasz-Piasecka1



Background The conventional form of chemotherapy including methotrexate are related with overall acute toxicity to healthy cells rapid elimination of chemotherapeutics from the body and low specificity towards target cancer cells Thus it was challenging to design a chemotherapeutic-carrier system overcoming these difficulties The HES-MTX nanoconjugate was obtained by covalent coupling of well-known therapeutic compounds ndash methotrexate as anticancer agent and hydroxyethyl starch as high-molecular carrier [1] The main advantage of HES-MTX nanoconjugate over the MTX in free form is prolonged half-time in plasma and specific biodistribution which is realized mainly by interacting with folate receptors alpha (FRα) overexpressed on tumor cells or through enhanced vascular permeability and retention effect (EPR)

The main objective of the study was to determine whether HES-MTX nanoconjugate can modulate the systemic antitumor immune response and affect the changes in the landscape of immune cells infiltrating into tissue of murine colon carcinoma MC38 This in turn should contribute to the creation of efficient immune response against growing tumor by DC-based vaccines multiple injected after chemotherapy administration

Material and Methods Female C57BL6 mice (8-10 weekd old) were subcutaneously inoculated with MC38 cells In course of chemotherapy on the

14th day of experiment mice received intraveno-usly MTX or HES-MTX (20 mgkg bm) and 3 days later (17th day of experiment) 5 mice from each group were sacrificed and tumors and spleens were dissected Mice received chemotherapy on 14th day of experiment and on the 17th 24th and 31st day of experiment tumor antigen-stimulated dendritic cell based vaccines (DCTAg) were applied peritu-morally In group of mice from untreated and chemotherapy treated groups tumor nodules and spleens were dissected on the 31st day of experiment and from DCTAg-receiving groups group tumor nodules and spleens were dissected on the 35th day of experiment (3-5 mice per group) Tumor cells and spleen cells were analysed by multipa-rameter flow cytometry according to the procedure described previously [2] During analyses the percentage of lymphoid as well as determination of their activity and percentage of myeloid cells as well as identification of macrophage stage polari-zation through expression of CD206 intra-cellular antigen were evaluated Moreover determination the ability of splenocytes obtained from treated MC38 bearing-mice to generate efficient anti-tumor response was conducted After five-days restimu-lation of spleen cells with mitomycin C-treated MC38 cells the phenotype and CD107a degranulation assay and cytotoxic activity of splc against MC38 cells were determined

38

Results

Three days after HES-MTX administration

the enlargement of CTLs population size

CD8+ T cells NK NKT cells) in spleen and

tumor tissue as well as in reduction of the

population size of immune cells with

suppressor activity (Tregs TAMs Mfs and

M2-type macrophages) in tumors

Moreover after restimulation spleen cells

obtained from HES-MTX-treated mice

revealed higher percentage of CD8+ T cells

and the highest cytotoxic activity of spleno-

cytes against tumor cells This contributed

to creation of favourable environment

necessary to promote the development of

anti-tumor immune response by dendritic

cell-based immuno-therapy which was

reflected especially in delay of tumor

growth


The administration of HES-MTX influenced

the systemic immune response and enhanced

the cytotoxic activity of splenic CTLs but

also changed the hostile landscape of immune

cells infiltrating into tumor tissue This

contributed to creation of favourable

environment necessary to promote the deve-

lopment of anti-tumor immune response by

dendritic cell-based immunotherapy which

was reflected especially in delay of tumor

growth

The study was funded by National Science

Centre (project no 201519NNZ602908

and 201727BNZ602702)

References [1] Goszczyński TM et al Pharma Res Per 2(3)

2014 e00047

[2] Rossowska J et al J Exp Clin Cancer Res 2018

37 126 doi 101186s13046-018-0799-y


Session 1 O 5

Regulation of mitochondrial dynamics and mitophagy

in 2-methoxyestradiol-mediated osteosarcoma- and glial-cell death

Paulina Przychodzen1 Alicja Kuban-Jankowska

1 Anna Olewniak-Adamowska

2

Michal A Zmijewski2 Dagmara Jacewicz

3 Zbigniew Kmieć

2 Agata Ploska

45

Aleksandra Dabrowska3 Stephan Nussberger

6 Leszek Kalinowski

46 Magdalena

Gorska-Ponikowska167

1Department of Medical Chemistry Medical University of Gdansk 2Department

of Histology Medical University of Gdansk 3Department of Chemistry University

of Gdansk 4Department of Medical Laboratory Diagnostics Medical University of Gdansk 5Biobanking and Biomolecular Resources Research Infrastructure Poland (BBMRIPL) 6Department of Biophysics Institute of Biomaterials and Biomolecular Systems University

of Stuttgart 7Euro-Mediterranean Institute of Science and Technology Palermo Italy

Background

Osteosarcoma (OS) is one of the most

malignant tumors of childhood and adoles-

cence [1] whereas gliomas are comparatively

rare human cancers of the central nervous

system (CNS) [2] Research on mitochondrial

dynamics (fusionfission) mitochondrial bio-

genesis and mitophagy have received much

attention in last few years as they are

crucial for understanding of many biological

processes including cancer cell death [1]

Specifically it was shown that increasing

the cytoplasmic dynamin-related protein

39

1 (Drp1) expression activates mitochondrial

fission which resulted in BAX activation

and downstream intrinsic apoptosis effect-

tively inhibiting osteosarcoma growth [1]

Notably Drp1 mediates also activation of

mitophagy specific type of autophagy [3]

2-methoxyestradiol (2-ME) is a physio-

logical derivative of 17β-estradiol that

possesses anticancer activities confirmed

by in vitro and in vivo studies [45]

Previously we evidenced that from mecha-

nistic point of view one of anticancer mode

of action of 2-ME is a selective induction of

overexpression of neuronal nitric oxide

synthase (nNOS) and enzyme nuclear

translocation Nuclear translocation of

nNOS results in local nitro-oxidative stress

leading to DNA damage in cancer cells [6]


We used highly metastatic osteosarcoma

143B and glial SW 1088 cell lines in the

study and cellular biology methods In order

to search for signaling pathways of 2-ME

we used flow cytometry stopped flow

electron microscopy and confocal micro-

scopy techniques

Results

Herein we present that nuclear generation

of nitric oxide leads to inhibition of mito-

chondrial biogenesis in highly metastatic

osteosarcoma 143B cells We further invest-

tigated whether 2-ME may be a regulator

of mitochondria dynamics in osteosarcoma

cell death model We demonstrated an

important role of mitochondrial division

in efficacy of 2-ME We also established

that the mitochondrial division inhibitor 1

MDIVI-1 induces cell death and sensitizes

the osteosarcoma cells to 2-ME-meditated

cell death


Herein we established that MDIVI-1 induces

cell death and sensitizes the osteosarcoma

cells to 2-ME-meditated cell death Pre-

viously it was reported that MDIVI-1

enhanced cisplatin induced cytotoxicity

in association with increased oxidative

stress [7] Indeed it was previously well

established that MDIVI-1 downregulates the

Drp1 [8] We observed that 2-ME upre-

gulated Drp1 level Due to induction of

nitric oxide derivatives we suggest that

2-ME-mediated S-nitrosylation of Drp1

may be one of the key regulatory adap-

tations for mitochondrial dynamics in

cancer For further investigation we will

explore role of mitochondrial dynamics in

osteosarcoma progression and in glioma

References [1] Jackson M Serada N et al PLoS

One13(12)e0209489 2018 doi 101371journal

pone0209489

[2] Fischer I Gagner J-P et al Brain Pathol

15(4)297-310 2006 DOI101111j1750-

36392005tb00115x

[3] Ikeda Y Shirakabe Aet al Circ Res 116(2)

264-78 2015 doi 101161CIRCRESAHA

116303356

[4] Kumar BS Raghuvanshi DSet al Steroids

1109-34 2016 doi 101016jsteroids201603017

[5] Kulke MH Chan JA et alCancer Chemother

Pharmacol 68(2)293-300 2011 doi

101007s00280-010-1478-7

[6] Gorska M Kuban-Jankowska A et al

Oncotarget 6(17) 2015 DOI1018632

oncotarget3913

[7] Tusskorn O Khunluck T et al Biomed

Pharmacother 111109-18 2019 doi

101016jbiopha201812051

[8] Manczak M Kandimalla R Yet al Hum Mol

Genet 28(2)177-99 2019 doi 101093hmg

ddy335


40

Session 1 O 6

Alterations in plasma concentration and activity of superoxide

dismutases in context of obesity andor type 2 diabetes

Łukasz Lewandowski1 Iwona Urbanowicz

2 Marta Kepinska

1 Halina Milnerowicz

1

1Department of Biomedical and Environmental Analyses Faculty of Pharmacy Wrocław

Medical University 2Department of Medical Laboratory Diagnostics Faculty of Pharmacy

Wrocław Medical University

Background

Superoxide dismutases (SODs) are a family

of enzymatic antioxidants Each SOD iso-

zyme originates from different cellular

compartments cytosol (SOD1) mitochondria

(SOD2) and extracellular fluidcell mem-

branes (SOD3) Lately and increasing level

of attention has been drawn to the activity of

SODs in context of insulin resistance as

SODs have been shown to protect pancreatic

beta cells from oxidative stress This research

aims to point out the possible effect of

obesity andor diabetes and other selected

factors on the concentration andor activity

of SODs


This research focuses on two often co-

existing diseases ndash obesity and type 2

diabetes The research aimed to examine the

concentration of SODs SOD1 SOD2

SOD3 and their corresponding activity

(total SOD activity CuZn-SOD activity

Mn-SOD activity) and compare these

values to total antioxidative capacity (TAC)

and the concentration of malondialdehyde

(MDA) a lipid peroxidation product These

variables were assayed for plasmaserum

obtained from 117 individuals of which

50 44 and 23 belonged to control group

obese non-diabetic group and diabetic

group respectively

Xanthine oxidase method was used to

measure total SOD activity in plasma

Potassium cyanide a selective CuZn-SOD

inhibitor was used in this method to

determine both CuZn-SOD and Mn-SOD

activity Plasma concentration of SOD1

SOD2 SOD3 were assayed for with use of

ELISA kits TAC was assayed for in uric

acid equivalents with use of copper

reduction method MDA concentration was

assayed for with use of thiobarbituric acid

method In this research the concentration

and activity of SODs were presented and

analysed in different units found in the

literature

Additional categorical factors analysed in

this research except for obesity and type 2

diabetes were sex and exposition to

cigarette smoke The exposition to cigarette

smoke was evaluated with use of an uni-

variate logistic regression model based on

the serum concentration of nicotine metabolite

ndash cotinine (assayed for with use of an

ELISA kit)

Results

Regardless of sex and exposition to cigarette

smoke the diabetic groups (non-obese and

obese) showed a markedly increased

concentration of SOD1 An increase in

CuZn-SOD activity was found in diabetic

groups compared to non-diabetic However

when displayed in [Umg SOD1+SOD3]

the values of CuZn-SOD showed a decre-

asing trend (control gt obese non-diabetic gt

diabetic non-obese gt diabetic obese)

Similar but less pronounced differences

were found in values of total SOD activity

but not in Mn-SOD activity Obese indivi-

duals were characterized of higher TAC

values than the non-obese

41

Regardless of obesity andor type 2 diabetes males were characterized of significantly higher SOD1 concentration TAC values were also higher in males compared to females although this difference was not significant in the obese non-diabetic group However no significant difference was observer in other analysed variables

There were no significant differences between the individuals exposed to cigarette smoke in all of the analysed variables Interestingly this lack of dependency was observed also in concentration of MDA

Discussion and conclusions This research featured both the values of different SODs in plasma simultaneously in the same dataset To our knowledge not many research papers cover such data thus ndash the discussion might prove difficult However several research showed a signi-ficant positive correlation of SOD activity with BMI or insulin [1 2] However some research show no such dependence [3]

Several research show higher SOD activity in obese compared to non-obese [4] and type 2 diabetic individuals compared to non-diabetic[5] Interestingly to our know-ledge an increase in plasma SOD1 concen-tration was not observed before in the diabetic group although an increase in SOD3 concentration has been observed

The results of this research may indicate that in case of obesity and type 2 diabetes the organism may adapt to increased oxidative stress with increase in TAC and CuZn-SOD activity

References [1] S Sfar R Boussoffara et al Nutr J 201312 DOI 1011861475-2891-12-18 [2] B Guadalupe B Duarte et al Metab Syndr Relat Disord vol 14 3 2016 DOI 101089met20150088 [3] F Erdemir D Atilgan et al Actas Urol Esp (English Ed vol 36 3 2012 DOI 101016jacuroe201205003 [4] O Erdeve Z Siklar et al Biol Trace Elem Res vol 98 3 2004 DOI 101385BTER983219 [5] HM Turk A Sevincet al Acta Diabetol vol 39 3 2002 DOI101007s005920200029


Session 2 O 1

Effect of the surface charge on the structure of lysosome adsorbed on

the gold surface

Paulina Komorek1 Izabella Brand

2 Barbara Jachimska

1

1Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences Krakow Poland 2Department of Chemistry Carl von Ossietzky University of Oldenburg Oldenburg Germany

Background The neurodegeneration process is associated with proteins which transform from their native form to partially changed structure and finally to aggregates which accumulate in affected tissues The progression of neuro-degeneration is associated with the growth of protein aggregated to fibrils It is preceded by initial changes in the secondary structure of the protein Adsorption of protiens on the

liquidsolid interface is known to be a fundamental phenomenon that can cause protein folding disorders An in-depth investigation of the effect of the surface-protein interactions on its secondary struc-ture could provide new insights into the aggregation mechanisms The aggregation of proteins in bulk solution has been exten-sively studied while in situ studies of secon-dary structure of proteins in the adsorbed state are stil not well understood

42

Material and Methods In the studies presented here lysozyme (L6876 Sigma Aldrich) from chicken egg white was used It was used without further purification Lysozyme adsorption on the gold surface as a function of the pH the electrolyte solution wass monitored by Multi-Parametric Surface Plasmon Resonance (MP-SPR) and Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) The secondary structure of lysozyme in solution was analyzed by Circular Dichroism (CD) while changes in the protein structure in the adsorbed state were investigated using Polarisation Modulation-Infrared Reflection Absorption Spectroscopy (PM-IRRAS)

Results In the presented work we selected hen egg lysozyme and gold surface to study factors having a major impact on the mechanism of protein adsorption and the conformation of the adsorbed protein on the solid surface By using MP-SPR and QCM-D the nature of the interactions and related amount of adsor-bed protein were investigated and allowed us to determine the orientation of molecules on the gold surface It was also confirmed that the rearrangement of molecules caused by changes in surface coverage displays themselves in the macroscopic properties of formed layers Comparison of CD and PM-IRRAS results in solution and in the adsor-bed state respectively showed changes in the secondary structures of lysozyme The adsorption of lysozyme was studied as a func-tion of surface charge on the protein

(varions of pH) and surface charge density on the metal surface The protein films were investigated using in situ PM-IRRAS Electrostatic attraction between the metal surface and lysozyme facilitate the protein accumulation on the Au surface The results of our studies show that in the protein misfolding process the appreance of disordered structures is first observed

Discussion and conclusions The presented data unambiguously indicate that protein surface coverage and charge at the surface of molecules can influence the properties and structure of molecules coming in contanct with solid surfaces The surface charge has a significant impact on the conformation and structure of proteins adsorbing on solid substrates However the mechanism of changes in the conformation of lysozyme at the surface is still not completely understood These results are particularly relevant for understanding the process of protein aggregation

Acknowledgements This work was partially supported by project NCN OPUS 201623BST502788 InterDokMed POWR030200-00-I01316 and NAWA PPNBIL2018100103

References [1] B Jachimska A Kozlowska et al Langmuir 28 11502-11510 2012 httpsdoiorg101021 la301558u [2] K Kubiak-Ossowska M Ćwięka et al Physical Chemistry Chemical Physics 17(37) 24070-24077 2015 httpsdoiorg101039C5CP03910J


Session 2 O 2

Enhancement of antimicrobial photodynamic therapy using metallic

nanoparticles

Ewelina Wanarska1

1Deparment of Organic and Medical Chemistry Wrocław University of Science

and Technology

43

Background

In XXI century bacterial infections are global problem The antibiotic therapy is the most common way of treatment The ESKAPE acronym describes bacteria that do not undergo antibiotic therapy Entero-coccus faecium Staphylococcus aureus Klebsiella pneumoniae Acinetobacter baumannii Pseudomonas aeruginosa and Enterobacter [1] Antimicrobial photody-namic therapy based on using non toxic dyes (photosensitizers) and special light in present of oxygen turned to be new approach to kill resistant bacteria by gene-ration of reactive species of oxygen [2] Nanotechnology as a field of study which deals with the creation and manipulation of metallic nanoparticles allows to create of new generation photosensitizers which are able enhance process of killness [3]


In this study the impact of various concen-trations of methylene blue and gold nano-particles was tested against Staphylococcus aureus using luminescence test and series of dilution method Then bactericidal effect of photodynamic inactivation with LED light irradiation was tested using series of dilution method Gold nanoparticles was used as enhancement agents of photody-namic therapy using previous mentioned method

Results

Methylene blue used as photosensitizer

caused 92 mortality of Staphylococcus

aureus cells after 30 minutes of irradiation

Gold nanoparticles used as enhancement

agents caused 96 mortality of Saureus

cells


Process of photodynamic therapy is widely

described Tawfik et al described similar

results using laser light irradiation (660 nm)

receiving 97 mortality of Saureus using

methylene blue and gold nanoparticles as

enhacement agents [4] Antimicrobial

photo-dynamic therapy using methylene

blue as photosensitizer caused bactericidal

effect The presence of gold nanoparticles

enhanced the antimicrobial effect of therapy

References

[1] H Boucher G Talbot et al Clinical Infectious

Diseases vol 48 1-12 2009 101086595011

[2] V Perez-Laguna L Perez-Artiaga et al

Frontiers in microbiology vol 8 1-9 2017

103389fmicb201701002

[3] D Tada M Baptista Frontiers in Chemistry

vol 33 2015 103389fmicb201701002

[4] A A Tawfik J Alsharnoubi M Morsy

Photodiagnosis and Photodynamic Therapy vol

12 215-220 2015 101016jpdpdt201503003


Session 2 O 3

Bile salts loaded Lipid Liquid Crystalline Nanoparticles for topical

administration of natural antioxidants

M Fornasier12

K Schilleacuten2 L Galantini

3 A Del Giudice

3 C Sinico

4 R Pireddu

4

S Murgia1

1 Department of Chemical and Geological Sciences University of Cagliari 2 Division of

Physical Chemistry Department of Chemistry Lund University 3 Department of Chemistry

Sapienza University of Rome 4 Department of Life and Environmental Sciences University

of Cagliari

44

Background

Bile Salts (BSs) are natural surfactants

deputed to lipid digestion in vivo The

behaviour of these biologically important

compounds has been well-studied to under-

stand many complicated biological systems

such as drug absorption in the small intestine

lipid bilayer mobility and phospholipid-bile

salts interactions [1-3] Nevertheless their

potential in nanomedicine for topical admi-

nistration has not been fully studied Indeed

the first layer of the skin stratum corneum

represents the first obstacle in this kind

of application due to its very dense and rich

lipid matrix in which the keratinocytes are

embedded Lipid formulations are able to

fluidify stratum corneum allowing a drug to

diffuse through it [4] Therefore we studied

the effect of BSs on Lipid Liquid Crystalline

Nanoparticles structure and their penetration

properties in vitro for the delivery of natural

antioxidants


Using monoolein as building block to

prepare NPs in water and Pluronic F108

as stabilizer the effect of three bile salts

(Sodium Cholate Deoxycholate and

Taurocholate) on the NPs physicochemical

features was studied After an initial

screening the best candidates in terms of

colloidal stability were loaded with catechin

a natural antioxidant and their penetration

properties on skin were evaluated in vitro

Results

SAXS data showed how BS affect mono-

olein self-assembly leading the system from

a cubic phase (cubosomes) to vesicular

structures When oleic acid is added to the

mixture a phase transition from unilamellar

vesicles to hexosomes is highlighted also by

Cryo-Tem (Fig 1)

DLS ELS and SAXS showed that Tauro-

cholate gave the most stable formulations in

comparison with Cholate and Deoxycholate

Catechin was encapsulated in hexosomes

BS-loaded hexosomes and vesicles and

these three formulations were compared in

vitro to understand how the structure and

BSs can affect the penetration properties


Among the BSs studied Taurocholate is the

most hydrophilic the presence of a negative

head-group (-SO3-) at the lipidwater interface

increased the NPs Zeta potentials giving

a higher colloidal stability to the aggregates

By tuning oleic acid and BS concentration

and therefore the curvature of the interface

it was possible to obtain vesicles or hexo-

somes The encapsulation of catechin did

not affect significantly the NPs physico-

chemical features The penetration tests in

vitro showed that the presence of BS in the

NPs can enhance the penetration properties

giving almost 26 of catechin in the first

layers of the skin (stratum corneum +

epidermis) in comparison with the hexosomes

and vesicles without BSs

In conclusion we investigate the effect of

BSs on the physico-chemical parameters of

NPs made with monoolein and stabilized by

Pluronic F108 Moreover we showed that

BSs are able to enhance significantly NPs

penetration properties on skin for topical

delivery of natural antioxidants

References [1] M Mohapatra and A K Mishra Langmuir vol

27 13461-13467 2011 DOI number 101021la

203028s

[2] J Gustafsson T Nylander et al Journal of

Colloid and Interface Science vol 211 326-335

1999 DOI number 101006jcis19985996

[3] K Schilleacuten L Galantini et al Physical

Chemistry Chemical Physics vol 21 12518-12530

2019 DOI number 101039c9cp01744e

[4] M Schlich M Fornasier et al Colloids and

Surfaces B Biointerfaces vol 171 675-681 2018

DOI number 101016jcolsurfb201808008

45

Fig 1 Cryo-TEM images of the samples containing Taurocholate


Session 2 O 4

Functionalization of curdlan gel for effective antibiotic bonding

Anna Michalicha1 Anna Belcarz

1

1Chair and Department of Biochemistry and Biotechnology Medical University of Lublin

Background

Currently various synthetic and natural

polymers are used for regenerative medi-

cine One of natural polymers of high

biomedical potential is curdlan which forms

hydrogel of high strength and elasticity

Also it exhibits high water-absorption and

retention capacity therefore may be useful

in fabrication of eg hydrogel dressing

materials or other biomaterial resources (Cai

and Zhang 2017)

Coupling of therapeutical substances to

curdlan matrix could increase its biological

potential However the disadvantage of

curdlan is the lack of active groups for

modifications such as amino and carboxyl

groups which could enable binding of

antibacterial or anti-inflammatory agents

Strategies used for curdlan functionalization

include among others sulfonation amination

oxidation esterification or phosphorylation

(Cai and Zhang 2017 Zhang and Edgar

2014) but they typically lead to curdlan

solubilization and loss of beneficial physic-

chemical properties Recently catecholamine

polymers were used for functionalization of

different matrices (Lee et al 2007)

Therefore catecholamine derivative was

used to functionalize the curdlan matrix by

formation of strongly adhesive ad-layer

Such functionalized matrix was used

as a template for antibiotic (gentamicin)

immobilization Some of the properties of

such produced drug-loaded hydrogel were

tested including antibacterial activity

against three reference bacterial strains and

drug release profile


8 (wv) curdlan suspension gelled at 93oC

was used as a matrix Functionalization of

polysaccharide matrix was performed by

hydroxytyramine polymerization from

2 mgml (or 4 mgml) solution for 24 h at

25oC Monomer (hydroxytyramine) was

added to curdlan matrix before its gelation

(sample 2-D-BG and 4-D-BG) or after

(2-D-AG) Non-attached polymer was eluted

from the matrix in DI water (as monitored

by UV-VIS spectrophotometry) Gentamicin

was immobilized on functionalized matrices

during their incubation in 1 mgml aqueous

drug solution with and without activation

with 5 glutaraldehyde Drug release from

the matrices was performed by incubation

46

of curdlan samples in PBS pH 74 at 37oC

with daily exchange of buffer In collected

samples gentamicin was estimated after

derivatization by phthaldialdehyde (Ginalska

et al 2004) Antibacterial activity of

functionalized curdlan was evaluated by

indirect method in extracts collected

in similar way as in drug release test (PBS

was replaced by Mueller-Hinton Broth)

The extracts were inoculated by S aureus

ATCC 25923 S epidermidis ATCC 12228

and E coli ATCC 25922 reference strains

and allowed to grow at 37oC for 24h Then

the bacterial growth was estimated as

optical density at 660 nm in Synergy H4

plate reader (Biotec USA) The water

uptake was evaluated during 72 hours

incubation in water as a function of wet

weight increase

Results

The catecholamine layer on matrix was

regular without noticeable precipitates and

colour differences Larger amount of

gentamicin was immobilized to the curdlan

sample which was modified by hydroxy-

tyramine added before polysaccharide

gelation (2-D-BG) in comparison with

added after gelation (2-D-AG) 2-fold increase

of amount of hydroxytyramine monomer for

curdlan functionalization caused the slight

(by approx 25) increase of drug immo-

bilization Activation with glutaraldehyde

did not affect the binding capacity of the

drug to functionalized curdlan matrix The

results of the experiment of gentamicin

release to PBS solution indicated that the

4-D-BG sample the most effectively released

the drug Determination of the antibacterial

properties of drug-loaded hydroxytyramine-

functionalized curdlan samples showed that

these matrices were able to protect the

culture medium against bacterial infection

for a long time The weakest protection was

observed against E coli strain while against

S epidermidis and S aureus strains this

protection lasted for a minimum 28 days

All samples absorbed water in 700-900

of its initial weight


Modification of curdlan matrix using poly-

hydroxytyramine allows efficient binding

and release of the antibiotic and protects the

matrix against bacterial infection The matrix

modified in this way can be used in future to

create dressing materials for the treatment of

postoperative wounds and to protect them

against infection

References [1] Cai Z and Zhang H Food Hydrocolloids vol

68 128-135 2017 101016jfoodhyd201609014

[2] Zhang R and Edgar KJ Biomacromolecules vol 15 1079-1096 2014 101021bm500038g

[3] Lee H Dellatore SM et al Science vol 318 426-430 2007 101126science1147241

[4] Ginalska G Osinska M et al Journal of

Biomaterials Applications vol 18 279ndash291 2004 1011770885328204041443


Session 2 O 5

Cytostatic effects of natural products on rhabdomyosarcoma

ndash in vitro studies

Weronika Kazimierczak1 Anna Szewczyk

12 Małgorzata Daczewska

1 Julita

Kulbacka2

1 Department of Animal Developmental Biology Institute of Experimental Biology Faculty

of Biological Sciences University of Wroclaw Sienkiewicza 21 50-335 Wroclaw Poland

2 Department of Molecular and Cellular Biology Wroclaw Medical University Borowska

211A 50-556 Wroclaw Poland

47

Rhabdomyosarcoma (RMS) is the most

common soft tissue sarcoma of the

childhood There are 3 types of RMS based

on histological diagnosis pleomorphic

rhabdomyosarcoma (PRMS) embryonal

rhabdomyosarcoma (ERMS) and alveoral

rhabdomyosarcoma (ARMS) Depending on

the type of the cancer it is located in soft

tissues of the extremities neck eye area or

genitourinary organs The treatment of RMS

invilves the radiation therapy and chemo-

therapy in combination with tumor resection

[1 2] and vincristine are currently approved

by the FDA (Food and Drug Administration)

for the treatment of RMS [3]

The aim of our research is to find new

potential cytotoxic substances against ERMS

We put emphasis on natural products such

as betulinic acid biochanine and jasplaki-

nolide The results of the MTT assay showed

that all tested substances are cytotoxic to

ERMS after 24 and 72 hours incubation

although the concentrations of the cytotoxic

substances differ from each other At the

same time the analysis of the effect of these

substances on fibroblasts was undertaken as

a control group for ERMS using the same

concentrations of the substances Each

substance is cytotoxic to fibroblasts but the

fibroblastsrsquo survival rate is different

compared to ERMS Optimal ERMS

cytotoxicity parameters will be selected

from the collected results which will be

used for further in vivo studies using the

zebrafish model

References

[1] P Balogh R Baacutenusz et al Diagnostic

Pathology vol 11(1) 99 2016 DOI

101186s13000-016-0552-9

[2] R Dagher L Helman The Oncologist vol

434-44 1999 DOI 101634theoncologist4-1-34

[3] httpswwwcancergovabout-cancertreatment

drugsrhabdomyosarcoma (access 13032013)


Session 3 O 1

Determination of genetically modified dendritic cellsrsquo ability

to survive migrate to the lymph nodes and infiltrate the tumor tissue

of MC38 colon carcinoma in in vitro and in vivo study

Jagoda Mierzejewska1

Katarzyna Węgierek1 Natalia Anger-Goacutera

1 Agnieszka

Szczygieł1 Magdalena Geneja

1 Bożena Szermer-Olearnik

1 Joanna Rossowska

1




Background

Dendritic cells (DCs) play crucial role in

regulation of the immune response They

are involved in promoting the humoral

response as well as cellular response [1]

Because of their huge potential DCs are

a promising tool in fight against cancer For

induction of specific immune response

research focusing on the application of DCs

led to the development of methods for their

ex vivo activation with tumor antigens

(TAg) Such stimulated DCs used as a vaccine

increase the probability of recognition of the

heterogeneity in cancer cell populations by

the host immune system and stimulation of

CD4+ and CD8+ T cells [12] However the

administration of DC-based vaccines leads

only to temporary reduction of tumor growth

For this reason their effect is enhanced by

delivery such cytokines as IL-12 andor IL-18

48

The aim of the study was to determine

genetically modified dendritic cells survival

and their ability to migrate to the lymph

nodes and to infiltrate the tumor tissue of

MC38 colon carcinoma


The C57BL6 mice were inoculated sub-

cutaneously (sc) in a right flank with

MC38 colon carcinoma cells (11x106

cellsmice day 0) On the 15th day mice

with established tumors were administered

peritumorally (pt) with BM-DCs geneti-

cally modified to IL-12 and IL-18 co-

production stimulated with MC38 tumor

cell lysate (2x106 cellsmice) or control

cells In order to identify transduced cells

in lymph nodes and tumor tissue staining

with a fluorescent dye CFDA-SE were

performed Lymphoid organs and tumor

tissue were collected from mice on the 3rd

5th and 7th day after a single administration

of cell vaccines In addition the prolife-

ration rate and survival time of vaccine cells

in vitro were evaluated

Results

In the first stage of research CFDA-SE

vaccine cell staining conditions were opti-

mized In the next stage of the in vitro

study the proliferation rate and survival

time of dendritic cells on the 3rd 5th and 7th

after a single administration of vaccines

were determined and were depended on the

type of gene transduction Transduced cells

stained with a fluorescent dye CFDA-SE

were observed in lymph nodes and tumor

tissue The numbers of CDFA-SE cells

identify in lymph nodes and tumor tissue

were depended on the type of applied vaccine

cells and the duration of the experiment

Discussion and conclusion

The obtained results suggest that DCs gene-

tically modified to overproduction of IL-12

andor IL-18 are able to survive proliferate

and migrate to the lymph nodes for 7 days

in in vitro and in vivo conditions and they

can be effective tools in anti-cancer therapy

This study was funded by National Science

Centre Poland (projects no 201517N

NZ402834 201727BNZ602702)

References [1] Steinman R M Banchereau J Nature vol

449419-26 2007

[2] Palucka K Banchereau J Nat Rev Cancer vol

12 265-277 2012


Session 3 O 2

Knowledge about coronary artery disease among Polish students

ndash survey study

Gabriela Kanclerz1 Karol Nowak

1 Jaroslaw Zalewski

2

1Studentsrsquo Scientific Group at the Department of Coronary Artery Disease and Heart

Failure Faculty of Medicine Jagiellonian University Medical College 2The Department of

Coronary Artery Disease and Heart Failure Jagiellonian University Medical College

Background

Coronary artery disease (CAD) is one of the

most common cause of death in Poland

Nevertheless the level of knowledge about

this pathology risk factors and complication

seems to be insufficient in our society The

aim of our study was to investigate the level

of knowledge about CAD among Polish

students


We conducted a survey study by internet

questionnaire Interviewees were 173

49

students of Polish universities The questio-

nnaire was prepared in accordance to

second version of Coronary Artery Disease

Education Questionnaire (CADE-Q II)

There were 31 questions that assess studentsrsquo

literacy in each four options to choose

right answer (for 2 points) half-right answer

(1 point) wrong and bdquoI donrsquot knowrdquo answer

ndash both marked as 0 points The maximum

overall score of the test was 62 points

Statistical analyses were performed with the

Statistica 131 (StatSoft Statistica 131

Tulsa Oklahoma USA) software Continuous

variables are expressed as a mean plusmn

standard deviation or median (interquartile

range) and categorical variables as a number

(percentage) Continuous variables were

first checked for normal distribution by the

Shapiro-Wilk test and then were compared

by Students t-test or U-Mann Whitney test

if distribution was normal or different than

normal respectively Categorical variables

were analyzed using the chi-squared test or

Fisherrsquos exact test All independent variables

associated (P lt02) with the score of questio-

nnaire in an univariate model and not corre-

lated with another independent variable

were then included in the multivariate linear

regression analysis to determine the score of

survey Two-sided P-value of less than 005

was considered statistically significant

Results

We collected answers from 173 participants

Among them there were 60 men (347)

The mean age of contributors was 220

(210-220) and the mean overall result of

the survey was 480 points (440-520) In

the questionnaire 118 participants declared

the contact with cardiovascular diseases

(CVD) that was defined as their own illness

or their family members or friends being

affected Surprisingly in direct comparison

of both groups ndash the students who had

contact with CVD and who not had there

were no significant differences in terms of

gender age the place of residence and the

sum of the survey The trend to higher self-

assessment of knowledge was observed in

contributors who had contact with CVD

(P=006) By multivariable analysis the

younger age (β=-087 P=0001 β ndash stan-

dardized linear regression coefficient) and

higher self-assessment of knowledge

(β=258 P=lt0001) was independently

associated with higher overall survey score


The knowledge about CAD in polish

students may be considered as insufficient

Unfortunately the personal contact with

CVD did not correlate with higher CAD

literacy Further CAD awareness campaigns

are necessary to gain adequate knowledge

about CAD in Polish students

References [1] Ghisi GL Grase SL Development and

psychometric validation of the second version of the

Coronary Artery Disease Education Questionnaire

(CADE-Q II) Patient Educ Couns 2015 98(3)

378-83 doi 101016jpec201411019


Session 3 O 3

Development and evaluation of microsponge based topical gel for acne

Sanjana Menezes2 Nabeel Asif Khan

1 Tabassum Khan

2 and Munira Momin

2

1Dept of Chemical Engineering University of Alberta Edmonton Canada 2SVKMrsquos Dr

Bhanuben Nanavati College of Pharmacy Mumbai Maharashtra India

50

Background Acne vulgaris is a common chronic skin condition caused due to inflammation of the pilosebeaceous unit ndash associated with the hair follicles and sebaceous glands Acne affects approximately 80 of teenagers and is characterized by tender inflammatory papules and nodules mainly scattered on the face neck chest and upper back The treatment of acne includes use of retinoids antibiotics herbal products anti-androgens vitamins and miscellaneous (salicylic acid and benzoyl peroxide) used alone or in com-bination

Novel drug delivery systems have been used to optimize the delivery of diverse therapeutic agents [1] Controlled release of the drug from the formulation into the epidermis such that the drug remains primarily localized with only a restricted amount of drug entering the systemic circulation is a means of controlling the side effects There is need to maximize the time for the active ingredient to remain on the skin while mini-mizing percutaneous transdermal absorption Recently several reports are published describing various drug loaded microsponge formulations of drugs for topical application Hence an attempt was made to develop a microsponge gel containing adapalene and azelaic acid for acne The objective of this study was to prepare microsponges using double emulsification solvent evaporation method study the effect of various formu-lation parameters like drug polymer ratio solvent polymer ratio stirring rate and emulsifier concentrations on the physical characteristics of the microsponges and study its efficacy using in vitro and ex vivo methods [2]

Material and Methods Azelaic acid and adapalene were selected as model drugs The microsponges were prepared by wow solvent evaporation method Factorial design was used to statistically optimize the formulation parameters The particles were evaluated for entrapment efficiency particle size scanning electron

microscopy (SEM) fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) The drug release from the microsponge loaded gel was studied using Franz diffusion cell Skin irritation of the developed formulation was studied on Albino wistar rats

Results The drug release study indicated that topical application of optimized gel enhanced the drug residence time in skin and therapeutic drug concentration was maintained up to 8 hrs The developed gel formulation was effective against acne causing bacteria [3]

Discussion and conclusion This study involved development of sustained release microsponge based gel for acne The microsponge system was optimized by appropriate variation of parameters like drug-polymer ratio volume of solvent (dichloromethane) stirring time and stirring speed The microsponges were incorporated into a suitable gel base It exhibited controlled release of azelaic acid and adapalene as compared to the marketed formulation Skin irritation study indicated the microsponges to be non-irritant Antibacterial study showed antibacterial activity of azelaic acid being retained on encapsulation The developed microsponges proved to be a suitable sustained release topical delivery system of adapalene and azelaic acid for acne better than the marketed conventional delivery systems

References [1] Chiara Sinico Liposomes as carriers for dermal delivery of tretinoin in vitro evaluation of drug permeation and vesicle ndash skin interaction Journal of Controlled Release 103 (2005) 123-136 [2] E Burchackaa et al New effective azelaic acid liposomal gel formulation of enhanced pharmaceutical bioavailability Biomedicine amp Pharmacotherapy 83 (2016) 771-775 [3] Nirav Patel et al Formulation and evaluation of microsponge gel for topical delivery of fluconazole for fungal therapy Journal of Pharmaceutical investigation 2016

51

Fig 1 SEM of optimized batch of microsponge


Session 3 O 4

Fluorescent polymeric nanocarriers of low cytotoxicity for two-photon

bioimaging

Katarzyna C Nawrot1 Urszula Bazylińska

2 Julita Kulbacka

3 Marcin Nyk

1

1Advanced Materials Engineering and Modeling Group Wroclaw University of Science and Technology 2Department of Physical and Quantum Chemistry Wroclaw University of Science and Technology 3Department of Molecular and Cellular Biology Wroclaw Medical University

Background With their strong photoluminescence narrow emission peaks [1] high quantum yields [2] and significant two photon absorption (TPA) cross sections [3] colloidal semicon-ductor nanoplatelets (NPLs) are promising candidates for bioimaging and photodynamic therapies However bioapplications are limited due to their toxicity [4] and hydro-phobicity which leads to aggregation of NPLs in biological liquids Here we present our method of preparation polymeric nano-carriers (NCs) in order to introduce hydro-philicity and lower toxicity of the inside-loaded semiconductor NPLs which does not change their optical properties

Material and Methods Polymeric NCs were prepared by vapori-zation of 55 ML CdSe NPLs dispersed in chloroform mixed with Pluronic 123 dichlo-rometane solution The resulting concentrate was stirred overnight with distilled water

Optical properties of the NPLs-loaded NCs ie optical density and photoluminescence was defined using spectroscopic methods TPA cross section was calculated using two-photon excited emission

Cytotoxicity of as-prepared material was determined using MTT assay on human gingival fibroblasts normal cell line and human ovarian cancer cell line

Results We obtained water-soluble spherical NPLs-loaded NCs of about 150 nm diameter with no significant change in photoluminescence signal or TPA cross section (up to 108 GM order of magnitude per one carrier) in wide range of wavelengths (670-1250 nm) in comparison to non-encapsulated NPLs Our method provides cells viability up to 95

Discussion and conclusions Preserved photoluminescence narrowness and high intensity as well as uniquely high

52

TPA cross section in the first biological transmission window [5] provide excellent optical properties for potential bioimaging application High hydrophilicity and significantly lowered toxicity allow to use the material in any biological environment while extended size of the NPLs-loaded NCs is a step towards selective take-up by mutated cells

References [1] S Ithurria M Tessier et al Nature Materials vol 10 936-41 2011 DOI 101038nmat3145

[2] Ithurria S Dubertret B Journal of the American Chemical Society vol 130 16504-5 2008 DOI 101021ja807724e [3] R Scott AW Achstein Nano Letters vol 15 4985-92 2015 DOI 101021acsnanolett5b00966 [4] N Chen Y He Biomaterials vol 33 1238-44 2012 DOI 101016jbiomaterials201110070 [5] N Won S Yeong Molecular Imaging vol 11 338-52 2012 DOI 1023107290201100057

Acknowledgements

KCN amp MN acknowledge support from the National Science Centre Poland under Grant no UMO- UMO-201829BST402172


Session 3 O 5

Mixture of MMP-2 MLCK and NOS inhibitors induce

cardioprotection against myocardial ischemiareperfusion injury

Anna Krzywonos-Zawadzka1 Agnieszka Olejnik

1 Marta Banaszkiewicz

1 Iwona

Bil-Lula1

1Department of Medical Laboratory Diagnostics Division of Clinical Chemistry and Laboratory Hematology Wroclaw Medical University Wroclaw Poland

Background After myocardial infarction (MI) a large volume of mechanical function of myocar-dium is lost It arises from heart injury due to generation of reactive oxygen species (ROS) during ischemia and postischemic organ reperfusion [1]

Nitric oxide (NO) is synthesized from L-arginine through a complex oxidation reaction catalyzed by NO synthase (NOS) and plays an important role in cardiovas-cular homeostasis The levels and bioactivity of NO are regulated by eNOS (endothelial NOS) nNOS (neuronal NOS) and iNOS (inducible NOS) as well as endogenous NOS inhibitors such as asymmetric dimethylarginine (ADMA) [2] During ischemiareperfusion (IR) a large amount of inducible nitric oxide synthase is produ-ced with subsequent increase of ADMA [3] ADMA disrupt nitric oxide signalling in endothelium by switch of the enzymatic activity from NO to ROS production In

consequence a toxic peroxynitrite (ONOO-) is formed that activates matrix metallo-proteinase 2 (MMP-2) MMP-2 mediates degradation of contractile proteins and nitratesnitrosylates of myosin light chain 1 (MLC1) and troponin I contributes to myocardial IR injury

The aim of this study was to verify if co-administration of subthreshold doses of doxycycline (MMP-2 inhibitor) L-NAME (non-selective inhibitor of NOS) and ML-7 (inhibitor of MLC phosphorylation by MLCK ndash myosin light chain kinase) regulates of NOS-ADMA-NO pathway leading to cardioprotection

Material and Methods Cardioprotective effect of the drug cocktail was tested on isolated rat hearts by Lan-gendorf method Hearts extracted from anesthetized male Wistar rats (300-350 g) were perfused with Krebs-Henseleit buffer after 25 min of aerobic stabilization hearts were subjected no-flow ischemia (20 min)

53

in the presence or absence of inhibitors mixture (Doxy (10 microM) ML-7 (05 microM) and L-NAME (2 microM)) followed by 30 min of aerobic reperfusion Next to hemo-dynamic parameters (coronary flow heart rate left ventricular developed pressure) biochemical markers of IR injury were measured in a heart tissue and coronary effluents

Results Mixture of Doxy (10 microM) ML-7 (05 microM) and L-NAME (2 microM) increased heart function at 85 of aerobic control The co-administration of subthreshold doses of inhibitors led to reduction of iNOS (plt0001) and ADMA levels to the level approximate to aerobic control (plt0002) and in turn increase in NO content to the level close to the aerobic control (plt0003) Additionally the positive correlation between iNOS and ADMA was found (r=088 p=0004) Level of both iNOS and ADMA negatively correlated with NO content (r= -083 p=0009 and r= -096 p=0001 respectively)

The activity of MMP-2 in cardiac tissue of rats subjected to IR was significantly higher compared to aerobic controls Co-admi-nistration of subthreshold doses of inhibitors led to normalization of MMP-2 activity to the level of aerobic control (plt0005) There

was a positive correlation between MMP-2 and iNOS (r=081 p=0008) as well as MMP-2 and ADMA (r=078 p=002)

Discussion and conclusions Thanks to synergistic effect of drugs the multidrug therapy with the subthreshold doses allows to address a few pathways of IR injury simultaneously and to achieve protection of cardiac function during IR This study confirmed that co-administration of subthreshold doses of Doxy ML-7 and L-NAME serves cardioprotective Additio-nally this study provided an important insight into understanding the interaction of iNOS eNOS and ADMA which is crucial for development the therapy beneficial for patients after myocardial infarction

This work was supported in part by the National Science Centre grant number UMO‐201623BNZ303151

References [1] IE Blasig S Shuter et al Free Radic Biol Med vol 16 35-41 1994 DOI number 1010160891-5849(94)90240-2 [2] Y Zhao PM Vanhoutte et al Journal of Pharmacological Sciences vol 129 83-94 2015 DOI number 101016jjphs201509002 [3] X Liu L Hou et al Nitric Oxide - Biology and Chemistry vol 54 73-81 2016 DOI number 104155fso1554


Session 3 O 6

Personalization of pancreatic cancer treatment- electroporation

electrochemotherapy and calcium electroporation pilot study

Julia Rudno-Rudzińska1 Julita Kulbacka

2 Maciej Guziński

3 Maciej Płochocki

4

Wojciech Kielan1

1Department of General and Oncological Surgery Medical University Hospital Borowska 213 50-556 Wrocław Poland 2Department of Molecular and Cellular Biology Faculty of Pharmacy with Division of Medical Analytics Wroclaw Medical University ul Borowska 211 50-556 Wrocław Poland 3Department of Radiology Medical University Hospital Borowska 213 50-556 Wrocław Poland 4Department of Oncology Medical University Hospital Borowska 213 50-556 Wrocław Poland

54

Background Pancreatic cancer has a very poor prognosis Despite of the development of cancer biology knowledge radiology cancer treatment all over the world this cancer has the same high mortality to morbidity index from years

The purpose of the study was to investigate safety of electroporation electrochemotherapy and calcium electroporation in pancreatic cancer treatment Qualification of the patients (therapeutic moment) safety and complications after procedures were examined

Material and Methods The group of 12 patients in this pilot study was operated in 2-nd General and Onco-logical Surgery Medical University of Wrocław Poland Main inclusion criteria were pancreatic cancer in all stages and recurrent disease Patient underwent electro-poration (IRE ndash irreversible electroporation) or electrochemotherapy (ECT ndash electroche-motherapy) with intravenosus admission of cisplatin or electroporation with calcium intratumoral administration

There was 1 patient with resectable pancreatic cancer 8 with non- resectable locally advan-ced pancreatic cancer 1 with metastatic disease and 2 with recurrence In 4 patients only electroporation was administrated 4 were with intratumoral calcium ions admi-nistration 2 with intravenosus cisplatin and 2 with both calcium and cisplatinum admi-nistration

Results The surgical and anaesthesiological procedure was safe There were 2 postoperative complications bile infection connected to the surgical by- pass procedure and mild pancreatitis connected to the IRE procedure with calcium ions administration There was also one 30-th day death because of circular insufficiency and fragile syndrome according to patient with oligometastatic disease

Discussion and conclusions Electroporation procedure is safe for enchancement of the surgical procedure and for patients with non-resectable locally advanced pancreatic cancer (LAPC) Patients with metastatic disease require caution in qualification because of fragile syndrome

Intravenosus cisplatin administration is safe and calcium ions intratumoral administration requires further investigation

The best therapeutic moment for each patient should be considered separately with interdisciplinary group of study

These are only pilot studies and the results are only observation rather than guidelines Also the sufficiency of these methods to the overall survival (OS) disease free survival (DFS) and progression free survival (PFS) needs to be continued because of too short time of observations

References [1] Martin CG et al Treatment of 200 locally advanced (stage III) pancreatic adenocarcinoma patients with irreversible eletroporation Annals of Surgery 2015 486-49 [2] Lambert L Et al Treatment of locally advancd pancreatic cancer by percutaneous and intraoperative irreversible electroporation general hospital cancer center experiance Neoplasma 2016 632 [3] Jaroszeski MJ Illingworth P Pottinger C Hyacinthe M Heller R Electrically mediated drug delivery for treating subcutaneous and orthotopic pancreatic adenocarcinoma in a hamster model Anticancer Res 199919((2A))989-94 [4] Granata V et al Electrochemotherapy in locally advanced pancreatic cancer Preliminary results International journal of surgery 2015 Jun18230-6 doi101016jijsu201504055 [5] Miklavcic D et al Electrochemotherapy from the drawing board into medical practice Biomedical engineering online 2014 Mar 1213(1) 29 doi1011861475-925X-13-29 [6] Hansen EL et al Dose dependent ATP depletion and cancer cell death following calcium electro-poration relative effect of calcium concentration and electric field strength PLoS 2015 Apr 810(4) e0122973 doi 101371journalpone0122973


55

Session 4 O 1

Thiazole based derivatives as sirtuins inhibitors Molecular docking study

Żaneta Czyżnikowska

zanetaczyznikowskaumedwrocpl Department of Inorganic Chemistry Wroclaw Medical

University 50-556 Wrocław Poland

Background

Sirtuin subtypes have been identified in

mammalian cells which are responsible for

the regulation of cellular metabolism trans-

cription and differentiation processes

Through a large number of their substrates

sirtuins are able to determine the biological

life expectancy including mitosis apoptosis

DNA repair and metabolism [1] They are

assigned to the III class of histone deace-

tylases but are able to catalyze the deacyla-

tion of various non-histone reagents

Therefore human sirtuins represent thera-

peutic target for the treatment of metabolic

dysfunctions neurological disorders age

related conditions and cancer [2] To date

many different potential inhibitors of

sirtuins have been described [3] Unfortu-

nately most of them show average rather

small potency and low selectivity There-

fore it is extremely desirable to develop

a new more potent and highly selective

compounds It is widely known that drug

discovery is time consuming and expensive

process The application of computational

techniques in design and optimization of

novel compounds allows to omit the tradi-

tional procedure and reduce time and cost of

drug development process


Within the presented project several

computational methods were used in order

to design the new compounds based on

thiazole scaffold We applied the structure-

based drug design approach where the

information about the three-dimensional

structure of molecular target is important

The geometries of analyzed derivatives

were optimized based on density level

theory Molecular docking simulation was

performed in order to obtain the binding

mode of proposed inhibitors towards sirtuin-

2 Additionally the qualitative and quanti-

tative analysis of intermolecular interactions

was performed

Results

The results obtained during molecular

docking revealed that all investigated

compounds are able to bind to the active

center of sirtuin The most stable complex is

characterized by the lowest binding free

energy (-326 kJmol) and also the lowest

inhibition constant (194 M) As can

be observed in this case the aromatic rings

of the ligand were exposed to hydrophobic

amino acid residues mainly Phe96 Leu103

Phe119 Ile169 Phe190 Val233 and Leu

239 (See Fig 1) There is also possibility of

H-bonding interactions involving Arg97 and

Val233 residues


The analysis of the issues presented here is

an important contribution to the problem of

design of selective inhibitors of sirtuins We

characterized in details the binding mode

of thiazole derivatives in the binding site of

sirtuin-2 what is a further step towards

better understanding the molecular reco-

gnition process and mechanism of inhibition

According to the previous studies the

proposed compounds might be involved in

interactions inside the induced hydrophobic

pocket similar to myristoylated-lysine

substrates [4] All analyzed complexes are

stabilized mainly by hydrophobic and van

der Waals forces Moreover the inhibition

56

constant allowed to predict the ability

of proposed ligand to inhibition of protein

and might be correlated to the half-maximal

inhibitory concentration Finally the

proposed derivatives may be used as lead

compounds for drug development to further

studies

Acknowledgments

Authors gratefully acknowledge the allotment

of the CPU time in Wroclaw Center of

Networking and Super- computing (WCSS)

References [1] M Schiedel D Robaa et al Medicinal

Research Reviews vol 38 147-200 2018

101002med21436

[2] Y Wang J He et al European Journal of

Medicinal Chemistry vol 161 48-77 2019

101016jejmech201810028

[3] X Bai L Yao et al Mini-Reviews in Medicinal

Chemistry vol 18 1151-1157 2018 102174

1389557516666160620095103

[4] LLYang HL Wang et al European Journal

of Medicinal Chemistry vol 155 806-823 2018

101016jejmech201806041

Figure 1 The binding mode of the most potent compound


Session 4 O 2

Fighting cancer with gravity

Dawid Przystupski1 Agata Goacuterska

2 Anna Szewczyk

2 Julita Kulbacka

2

1Faculty of Medicine Wroclaw Medical University 2Department of Molecular and Cellular

Biology Wroclaw Medical University

Background

Numerous studies have reported that gravity

alteration has remarkable influence on

growth and biological processes of human

cells Most studies have tended to focus on

the impact of altered gravity on human

cells however issues linked to malignant

cells have not been dealt with in depth

Therefore gravity-related experiments have

become an promising method to improve

our knowledge in the field of cancer biology

and may be useful to detect interesting

implications for future cancer treatment

Taking this concept further we studied the

effect of simulated gravity (sμg) on human

cancer cells using Random Positioning

Machine (RPM)


In an attempt to determine whether altered

gravity might be one of the factors modu-

lating multidrug resistance (MDR) in cancer

cells we used well defined commercial

57

human ovarian cancer cell line SKOV-3

resistant to cisplatin and doxorubicin The

cells were seeded on T25 cell culture flasks

fully filled with growth medium (without

the presence of air bubbles) and exposed to

simulated microgravity for 2h in the

presence of cisplatin as a model of cyto-

static drug administered directly before the

experiment After centrifugation the cells

were detached and seeded on 6-well and 96-

well plates for 24 and 72 hours to perform

cytotoxicity proliferation cell death and

cell cycle analyses Additionally the cells

were cultured on coverslips and fixed

directly after the centrifugation to evaluate

cell morphology using 3D Cell Explorer

(Nanolive) confocal and scanning electron

microscope

Results

Our studies revealed that SKOV-3 cells are

susceptible to simulated microgravity which

affects cell morphology and drug efficiency

We observed altered cell shape presence of

membrane blebbing lack of lamellipodia

and intracellular rearrangement of cyto-

skeletal fibres (actin β tubulin and zyxin)

even when the cells were cultured on RPM

for 2 hours (Fig 1) Cytotoxicity and cell

death assays showed increased percentage

of apoptotic cells after centrifugation on

RPM in the presence of cisplatin in com-

parison to control not centrifuged cells

Additionally clonogenic and cell cycle

assays revealed decreased percentage of

proliferative cells and G1G0 arrest

Conclusions

We believe that gravitational stress may

affect cell pathways involved in multidrug

resistance phenomena especially associated

with cell membrane and cytoskeleton

resulting in higher sensitivity of cancer cells

to chemotherapeutics The investigation and

clarification of these phenomena may

constitute initial step toward enhancing our

understanding of the relationship between

cellular resistance to chemotherapy and the

response to various gravitational stimuli In

our view this experiment constitutes an

excellent initial step toward enhancing our



response to gravity alteration

Figure 1 SKOV-3 cells morphology evaluated using 3D Cell Explorer microscope after culturing

for 2h on RPM


58

Session 4 O 3

Combination of EP and PDT in melanoma treatment

Wojciech Szlasa1 Olga Michel

2 Karolina Cierluk

3 Aleksander Kiełbik

1 Stanisław

Supplit1 Mounir Tarek

4 Anna Szewczyk

25 Jolanta Saczko

2 Julita Kulbacka

2

1Faculty of Medicine Wrocław Medical University 2Department of Molecular and Cellular

Biology Faculty of Pharmacy Wroclaw Medical University 3Faculty of Chemistry

Wrocław University of Science and Technology 4Universiteacute de Lorraine CNRS LPCT

F-54000 Nancy 5Department of Animal Developmental Biology Institute of Experimental

Biology University of Wroclaw

Background

Curcumin is widely known for its high

potency as an anticancer drug [1] Due to its

hydrophobic properties it seems to be

especially effective towards cutaneous and

subcutaneous tumors Nowadays more and

more effort is being devoted to enhance

cytostatic properties of the drug with the use

of novel therapies such as photodynamic

therapy (PDT) and electrochemotherapy

(ECT) Both of them show promising

effects when applied alone but recent data

suggest that their combination can be

beneficial [2]

In this project the authors propose a pro-

tocol for effective combination of PDT and

ECT validated by a set of experiments


The experiments have been performed on

melanotic (A375) and amelanotic (C32) cell

lines while fibroblasts have been used as

a model of non-cancerous cells To study

the PDT and ECT protocols we analysed

the effects of irradiation and of high electric

fields on curcumin using mass spectrometry

methods Immunofluorescence staining stu-

dies as well as viability tests were performed

on all cell lines The interaction of curcumin

and its derivatives with model cell mem-

branes namely lipid bilayers was studied

using molecular dynamics simulations

Results

Our analyses show that during PDT cur-

cumin undergoes decomposition to more

potent and smaller compounds such as

vanillin and ferulic acid In ECT on the

other hand curcumin loses sequentially its

methoxy groups Due to its rather hydro-

phobic nature curcumin first partitions

within the lipid membranes (cells envelop)

With time it changes its localization to

intracellular membranes

Overall concerning the effectiveness of

using curcumin as anticancer agent the

preincubation with curcumin has led to

much worse results


Two hypotheses can explain the obtained

results (1) either irradiation of the photo-

sensitizer disrupts the membranes in which

it localizes leading to extensive damage (2)

or inside the cells curcumin metabolism

being rapidly metabolized the effectiveness

of PDT is drastically reduced

At any rate the data we have gathered show

that the most effective way of combining

both therapies is to electroporate simulta-

neously after addition of the drug and

irradiate afterwards

Further studies are now required in order to

test whether these in vitro protocols to

effective successful cancer therapy

59

Acknowledgments

This research was supported financially by

the Subsidy Funds of Department of Molecular

and Cellular Biology SUBD26020009

The authors would like to thank dr Hanna

Czapor-Irzabek (Elementary Analysis and

Structural Studies Wrocław Medical

University Poland) for the assistance in

mass spectrometry analyses and expert

support

References [1] A Kielbik et al Effects of photosensitization

of curcumin in human glioblastoma multiforme

cells In Vivo (Brooklyn) vol 33 no 6 pp 1857-

1864 2019

[2] A Zielichowska et al The photodynamic effect

of far-red range phthalocyanines (AlPc and Pc

green) supported by electropermeabilization in

human gastric adenocarcinoma cells of sensitive

and resistant type Biomed Pharmacother vol 69

pp 145-152 Feb 2015


Session 4 O 4

Pulsed Current Evaluation for Prediction of Tumor Permeabilization

Rate

Veronika Malyško1 Vitalij Novickij

1 Augustinas Želvys

2 Austėja Balevičiūtė

2

Auksė Zinkevičienė2 Jurij Novickij

1 Irutė Girkontaitė

2

1Faculty of Electronics Vilnius Gediminas Technical University Vilnius Lithuania 2Department of Immunology State Research Institute Centre for Innovative Medicine

Vilnius Lithuania

Background

Electroporation is a phenomenon of increased

biological cell membrane permeability

facilitated by pulsed electric fields (PEF)

[1] One of the most established biomedical

applications of electroporation is electro-

chemotherapy when chemotherapeutic

agents are delivered to the tumour by

increase of cellular membrane permeability

[2 3] However during the clinical proce-

dures usually there is no feedback on the

efficacy of the treatment and the success

rate of the procedure is unknown until

several days post-treatment In this work we

tested the feasibility of pulsed current

measurements to serve as a real-time

indicator of successful tumour perme-

abilization by electric fields


BALBc mice were bred and housed in

mouse facility of State Research Institute

Centre for Innovative Medicine Vilnius

Lithuania 1times106 of SP20 myeloma cells

in phosphate-buffered saline (PBS) were

inoculated under the skin on the back of the

6ndash8 weeks old mice The tumors were

allowed to establish and grown until they

reached ~150-500 mm3 and were ready to

treat 12 mgkg of doxorubicin (Ebewe

pharma Austria) was injected intraperito-

neal 15-20 minutes prior to the treatment

Up to 3 kV 100 ns ndash 1 ms square wave high

voltage and high frequency (up to 1 MHz)

pulse generator was used for electroporation

Two electroporation protocols were employed

1) 14 kVcm x 100 micros x 8 pulses and 2)

35 kVcm x 800 ns x 1000 pulses Needle

electrodes with a gap of 5 mm were used for

pulse delivery

For in vitro experiments commercially

available electroporation cuvette with 1 mm

gap aluminum electrodes (Biorad Hercules

USA) was used and the parametric cell

permeabilization curve was acquired using

Propidium Iodide (PI 45 μM) (Sigma-

60

Aldrich Germany) and flow cytometry

(Amnis Seattle USA)

All experimental protocols were approved

by the Lithuanian State Food and Veterinary

Service (approval no 02-24) and carried out

in accordance with the the Guide for the

Care and Use of Laboratory Animals

Results

During microsecond pulse procedure the

pulses were delivered with a 30 s delay to

prevent any influence of Joule heating

A maximum 9 increase of current was

detected between the first and the last pulse

however the increase of current was statis-

tically significant only between the first and

(3-8 pulses) (Plt005 n = 3) To summarize

the current was increasing during the first 3

pulses followed by a saturation when higher

number of pulses was applied

Similar tendency was observed during

nanosecond pulsing procedure A distin-

guishable increase of the current between

the first and the second pulsing bursts was

detected The variation of current for all the

other pulse sequences (200ndash800 pulses) was

not statistically significant

In vitro data in the microsecond pulse range

indicated that the permeabilization rate of

the cells is saturated (gt95) after 4th pulse

which is in agreement with current increase

tendency In case of nanosecond protocols

the permeabilization rate is saturated after

the first 100 pulses which is also in

agreement with in vivo current measu-

rements

Both protocols triggered a statistically

significant tumor response to electrochemo-

therapy

Conclusion

It was shown that the changes in current can

serve as an indicator of electroporation The

result was confirmed both in vitro and

in vivo however the methodology is limited

to non-thermal PEF treatments

Acknowledgement

The research was funded by Research

Council of Lithuania Grant Nr S-MIP-19-

22

References [1] A Rolong R V Davalos and B Rubinsky

History of Electroporation in Irreversible Electro-

poration in Clinical Practice 2018 pp 13-37

[2] J Saczko et al The effectiveness of chemo-

therapy and electrochemotherapy on ovarian cell

lines in vitro Neoplasma 2016

[3] A Szewczyk J Gehl M Daczewska J Saczko

S K Frandsen and J Kulbacka Calcium

electroporation for treatment of sarcoma in

preclinical studies Oncotarget 2018


Session 4 O 5

A multifunctional nanoprobe based on polymeric nanocapsules loaded

with quantum dots and lanthanide doped nanocrystals

Magda A Antoniak1

Urszula Bazylińska2 Marcin Nyk

1

1 Advanced Materials Engineering and Modelling Group Wrocław University of Science

and Technology Wybrzeże Wyspiańskiego 27 50-370 Wrocław Poland 2 Department

of Organic and Pharmaceutical Technology Faculty of Chemistry Wrocław University

of Science and Technology Wybrzeże Wyspiańskiego 27 Wroclaw 50-370 Poland magdaantoniakpwredupl

61

Colloidal quantum dots (QDs) exhibit several numbers of unique size-dependent optical properties eg narrow band-gap emission and large absorption cross-sections Moreover they are interesting candidates for bioima-ging because of large one- and two-photon absorption cross-sections On the other hand inorganic upconverting nanocrystals can be used for designing new bio-medical markers due to good optical stability low toxicity and high signal-to-noise ratio of the up-conversion emission They are excited by near-infrared (NIR) radiation which can convert into visible emission The main advantages of NIR excited emission is high penetration depth through many materials including biological tissues and the possibility of applying relatively cheap and easily accessible continuous wave (CW) laser diodes

One primary problem with bioapplication of QDs and upconverting nanocrystals is their transfer to an aqueous solution To over-come this problem some approaches have been made including using biomolecules such as proteins as the capping agents for quantum dots [1] as well as encapsulation of the nanocrystals within a polymer[2]

For the purpose of designing multifunc-tional nanoprobe dispersed in water hydrophilization of colloidal CdSe QDs and NaYF4YbEr nanocrystals into hydrid structures was employed using encapsu-lation process of nanocomponents

In order to obtain nanocapsules with the most effective properties we prepared series of samples with different nanoparticles concentrations Firstly we studied optical properties of nanocapsules dispersed in water Next we investigated individual nanocapsules using scanning confocal fluorescence microscope equipped with piezo-electrically controlled sample holder and high NA oil-immersion objective The structure and morphology of samples were characterized with X-ray scattering tech-nique and transmission electron microscope measurements

We conclude that encapsulation method can combine the features of two kinds of nanocrystals into a single architecture and it is possible to obtain dual emission from two kind of nanocrystals (CdSe QDs and NaYF4YbEr at the same time) The deter-mined luminescence properties indicate that the NaYF4YbErCdSe QDs assemblies are efficient imaging agent dispersible in aqueous solution

Acknowledgements MA amp MN acknowledge support from the National Science Centre Poland under Grant no UMO- 201830EST500718

References

[1] MA Antoniak J Grzyb M Nyk Journal of Luminescence vol 209 57-60 2019 DOI number 101016jjlumin201901029 [2] U Bazylińska U Wawrzyńczyk D Kulbacka J et al Sci Rep vol 6 29746 2016 DOI number 101038srep29746

Fig1 Schematic graph of encapsulation processes of nanocomponents


62

Session 4 O 6

Light-induced in situ TEM microscopy revealing ultrastructural

interactions in antimicrobial photodynamic therapy

Andrzej Żak1 2

Marta Piksa2 Krzysztof Pawlik

2 Sylwia Nowak

3 Katarzyna

Matczyszyn4

1Electron Microscopy Laboratory Wrocław University of Science and Technology 2Ludwik

Hirszfeld Institute of Immunology and Experimental Therapy PAS Wrocław 3Laboratory of

Microscopic Techniques University of Wrocław 4Advanced Materials Engineering and

Modelling Group Wrocław University of Science and Technology

Background

The teams main research topic is the

development of antimicrobial photodynamic

therapy (aPDT) methods [1] and the deve-

lopment of appropriate lighting sources for

its purposes [2] The project described

below aimed at understanding the ultrastruc-

tural mechanisms that lead to the therapeutic

effect of aPDT


The basis for modification was Hitachi H-

800 microscope with a tungsten thermal

emission and high voltage of 200kV To

make the liquid cell sample containing

bacteria and photosensitizer the double 15

nm amorphous carbon on the copper grids

were used The in situ illuminator mounted

inside the TEM column contained 660 nm

LED emitter and applicable light pipe The

light intensity was calibrated to achieve 05

mWcm2 on the sample The microorganism

used for photodynamic therapy imaging and

survival tests was Staphylococcus aureus

The photosensitizer used during in situ

aPDT was a solution of methylene blue in

PBS at a concentration of 250 μgmL

Results

The method used allowed observation of

bacteria surrounded by a photosensitizer and

observations in a native hydrated state (fig 1)

Preliminary observations shown the morpho-

logical changes in the cell wall caused by

the generation of singlet oxygen during

PDT Dissection and disintegration of the

bacterial outer shell and peptidoglycan layer

allowed photosensitizer infiltration near the

cell membrane This lead to cell lysis and

bacterial death


The performed modification of the TEM

device shows that a certain group of

dynamical in situ biological observations

could be carried out in transmission emission

microscopes this means that the sample

does not need to be typically fixed and can

be influenced during observation This

requires a suitably short electron beam

interaction but offers the opportunity to

observe dynamic phenomena on a true

nanometric scale

The authors gratefully acknowledge funding

from the National Science Centre (PL)

under Miniatura grant number 201903

XNZ302100

References [1] Maliszewska et al Photochemistry and

Photobiology 93 (2017) 1081-1090 DOI

101111php12733

[2] Cheng et al npj Flex Electron 3 (2017) 18 DOI

101038s41528-019-0058-0

63

Figure 1 The exemplary TEM image of a prepared liquid cell and cross-section diagram


Session 6 O 1

Changes in nuclear proteome associated with lamin in the Drosophila

melanogaster model system after heat induction

Marta Pałka1 Aleksandra Tomczak

1 Jadwiga Jabłońska

1 Ryszard Rzepecki

1

1Faculty of Biotechnology University of Wroclaw

Background

One of the best examined extracellular

stressors is heat-shock induction Cells in

response to increased temperature have

developed an evolutionarily conserved

process- heat shock response (HSR) During

HSR the heat shock transcription factor

(HSF) binds to the promoters of hsp (heat

shock proteins) genes resulting in activation

of heat-inducible genes and a global down-

regulation of transcription Moreover it has

been found that after heat shock induction

the decondensation of chromatin occurs [1]

Changes in interaction between chromatin

and protein after heat shock were also

observed with major karyoskeletal proteins

involved in chromatin organization ndash lamin

It belongs to V-type intermediate filaments

exerting structural and regulatory functions

in the cell nucleus [2] Our hypothesis is

that lamins together with topoisomerase II

(Top2 is an enzyme required for DNA

regulations) may play a key role in

chromatin remodeling during HSR

For our studies we chose Drosophila mela-

nogaster as a model system due to the pre-

sence of only two lamin genes ndash B-type

(lam Dm) and A-type (lam C) and a single

isoform of HSF which makes it a definitely

simpler model than vertebrates In this

study we focused on investigating diffe-

rences between normal and heat shock

condition with regard to changes in protein

complexes associated with lamin Dm

together with post-translational modify-

cations which may be crucial in processes

occurred during HSR


All experiments were performed on

D melanogaster embryonic cell line ndash Kc

Cells were maintained in suspension culture

(in Schneider`s Drosophila Medium from

Gibco with 10 FBS and 1 antibiotics) at

23degC as normal conditions To induce the

64

heat shock cells were incubated at 37degC for

1 h before further experiments To identify

proteins interacting with lamin 1 PFA

cross-linking (10 min RT) followed by co-

immunoprecipitation (co-IP) under dena-

turing conditions (based on the protocol

from ThermoFisher dedicated to Pierce

Protein AG Magnetic Beads) Samples after

co-IP were next digest by FASP method

tryptic peptides were analyzed by tandem

mass spectrometry analysis (LC-MSMS)

MSMS data were processed using the

Mascot searching engine (UniProt Droso-

phila database combined with The common

Repository of Adventitious Proteins

cRAP)

Results We aimed to confirm the interaction between lamin Dm and topoisomerase II in both normal and heat shock conditions We observed extreme change in the number of proteins identified in MS after heat shock (almost 70 more interactors identified in comparison to control) After the classi-fication of identified proteins we observed changes in clusters in both groups based on protein functions In HS samples we observed an increased number of proteins involved in DNARNA binding Based on the quantitative analysis we showed about 30 decreased of lamC identifiers (the best-known interactor of lamDm q-value= 003)

30 increase of Top2 identification after hs (but the result is ns)

Discussion and conclusions Previous experiments suggest that lamin and topoisomerase II are involved in the regu-lation of transcription during heat shock induction and moreover they interact directly with chromatin We showed the interaction between them and along with other protein identifications from co-IP experiments its confirm us in this belief To determine whether the interaction is direct or indirect (through chromatin) further experiments have to be performed (co-IP with nucleic acid digestion) Changes in lamin- interacting proteome may be the result of the re-localization of lamin Dm after induction of heat shock or might be the effect of different phosphorylation rates in both conditions Observed protein pattern of interactors with lamin Dm after heat shock induction leads us to conclude that lamins may play a role in the epigenetic shutdown of transcription after heat shock-induced together with other components of a protein complex involved

References [1] D Strenkert et al The Plant Cell vol 23 2285-2301 2011 DOI 101105tpc111085266 [2] R Rzepecki et al Journal of Cell Science vol 111 121- 129 1998


Session 6 O 2

Antiviral activity of extract of Ginkgo biloba (EGb) and its phytochemical constituents against herpesviruses HHV-1 and HHV-2

Marta Sochocka1 and

Michał Ochnik

1 Maciej Sobczyński

2 Katarzyna Zwolińska

1

Egbert Piasecki1 Jerzy Leszek

3

1Laboratory of Virology Hirszfeld Institute of Immunology and Experimental Therapy Polish Academy of Sciences Weigla Street 12 53-114 Wroclaw Poland 2Department of Genomics Faculty of Biotechnology University of Wroclaw Fryderyka Joliot-Curie Street 14a 50-383 Wroclaw Poland 3Department of Psychiatry Wroclaw Medical University Wybrzeże L Pasteura 10 50-367 Wroclaw Poland

65

Background

Human alphaherpesvirus 1 (HHV-1) and

Human alphaherpesvirus 2 (HHV-2) belong

to the most common worldwide infections

of humans producing a lifelong infection

Several factors such as immune impair-

ment UV or stress lead to virus reacti-

vation in the place of the initial infection

(oral cavity lips or genital organs) causing

pain and skin ailments [1] Despite the

availability of several anti-herpesviral

agents it should be emphasized that the

need for new inhibitors is highly en-

couraged due to the increasing resistant viral

strains as well as complications linked with

periods of recurring viral replication and

reactivation of latent herpes infection [2]

The most promising are preparations of

a natural origin such as extract of Ginkgo

biloba (EGb) [3] as an alternative to com-

mercially available synthetic preparations

We evaluated the antiviral activity of EGb

and its phytochemical constituents flavonoids

and terpenes against HHV-1 and HHV-2


Standardized dry extract from G biloba

leaves mix of flavonoids and mix of

terpene lactones as well as single flavonoids

from G biloba isorhamnetin kaempferol

and quercetin were investigated Inhibition

of HHV-1 and HHV-2 replication was exa-

mined by early viral entry assay (inacti-

vation assay) Serial concentrations of EGb

and its phytochemical components were

incubated with HHV-1 or HHV-2 for 05 h

(Short-Term) 1h and 2h (Long-Term)

Viral titer was expressed with reference to

the TCID50 (tissue culture infectious dose)

value based on the cytopathic effects (CPE)

caused by the virus in approximately 50

of infected cells An appropriate Gompertz

growth model and exponential model were

fitted to estimate the concentration-depen-

dent decrease in virus titer after treatment

with EGb and its phytochemical components

Results

Pretreatment of the herpesviruses with EGb

mix of flavonoids mix of terpene lactones

and flavonoids from G biloba isorham-

netin kaempferol and quercetin prior

to infection of cells were studied EGb

produced a remarkable anti-HHV-1 and

anti-HHV-2 activity The extract affected

the viruses before adsorption to the cell

surface at non-cytotoxic concentrations

what is an important benefit of this extract

Even by 4 log TCID50 reduction of both

viruses titer with EGb was observed which

means a 9999 decrease in infectivity

Flavonoids from EGb especially isorham-

netin are responsible for the antiviral activity

of the extract Such activity was absent in

quercetin and kaempferol However EGb

showed the most potent antiviral potency

compared to isorhamnetin We have investi-

gated also an antiviral activity of EGb

against other viruses belonging to different

taxonomic groups such as Human adeno-

wirus 5 (HAdV-5) Vesicular stomatitis

virus (VSV) and Enteric cytopathogenic

bovine orphan virus (ECBO) EGb however

did not express antiviral activity against any

of these viruses A strong antiviral activity

of EGb was observed only for herpes-

viruses


Standardized EGb shows high anti-HHV-1

and anti-HHV-2 activity in non-toxic con-

centrations and significantly reduces the

infectivity of both pathogens Most likely

EGb could augment current therapies for

herpes labialis and genital herpes especially

in the treatment of skin ailments during

recurrent infections [4] A combination of

antiviral agents with different molecular

targets including EGb has the potential to

keep HHV-1 and HHV-2 replication

to a minimum

66

References [1] DZ Rechenchoski LC Faccin-Galhardi et al

Folia Microbiol 62 151ndash156 2017 DOI

101007s12223-016-0482-7

[2] SH James MN Prichard Curr Opin Virol

854-61 2014 DOI 101016jcoviro201406003

[3] TK Mohanta Y Tamboli et al Nat Prod Res

28 746-752 2014 DOI 10108014786419

2013879303

[4] M Sochocka J Leszek Patent Polski (16-09-

2019) Zastosowanie wyciągu z liści miłorzębu

japońskiego Ginkgo biloba (zgłoszenie nr P

423496 18-11-2017)


Session 6 O 3

Trimethylammonium 1-mercapto-1-carbadodecarnorate (TMA) ndash pharmaceutical precursor in BNCT method

Karolina Woacutejciuk1 Anna Bojanowska-Czajka

2 Ewelina Chajduk

2 Michał Dorosz

1

Grzegorz Woacutejciuk3 Michał Gryziński

4

1Radiological Metrology and Biomedical Physics Nuclear Facilities Operations Department National Centre for Nuclear Research 2Laboratory of Nuclear Analytical Methods Institute of Nuclear Chemistry and Technology 3Chemistry Department Central Forensic Laboratory of the Police 4Nuclear Facilities Operations Department National Centre for Nuclear Research

Background

Statistical data clearly indicate an increase

in the incidence of tumours every year

Probably that is a result of the changing

lifestyles and the progressive globalization

There are two factors crucial for the suc-

cessful treatment of the disease these are

the early detectability and the specificity of

the applied therapy We need new com-

pounds for the effective detection and

elimination of tumours Boron Neutron

Capture Therapy (BNCT) [1 2] selectively

targeted the tumour cells can be an effective

solution to this problem The therapy

belongs to the so-called bi-modal therapies

(1) a pharmaceutical containing molecules

that preferentially penetrate cancer cells and

are not absorbed (or little absorbed) by

healthy cells are administered to the patient

The molecules are labelled with boron

stable isotope 10B (2) the patients body is

irradiated with epithermal neutrons of some

specific energy Thermal neutrons are prefe-

rentially captured by boron nuclei Having a

neutron captured stable 10B nucleus trans-

forms into a meta-stable 11B nucleus(1)

which almost instantly decays along one of

the following paths (1)

Both particulate reaction products (7Li and

α-particle) transfer their energy into the

surrounding tumour tissue destroying her

Therefore the high accumulation and

selective delivery of 10B into the tumour

tissue are the most important requirements

to achieve efficient BNCT therapy Three

important parameters should be considered

in the development of boron carriers (1) the

boron concentrations in the tumour should

be in the range of 20-35 mg 10B per g (2)

the tumournormal tissue ratio should be

greater than 3-5 and (3) the toxicity should

be sufficiently low [1]

That the three principles above (1-3) be met

searching for new carriers of boron-10 is

both necessary and important [1 3] The

aim of this project is to characterize a new

carrier for clusters of boron-10 trimethyl-

ammonium 1-mercapto-1-carbadodecarnorate

(TMA) The selected chemical compound

has an analogous structure for clinically

used sodium mercaptododecaborate (BSH)

TMA has a -SH group that enables coupling

67

reactions with neurotransmitter proteins

(targeted therapy)

The aliphatic chain increases lipophilicity

facilitates crossing the blood-brain barrier

(brain cancer therapy)


In this work there were used a number of

chemical and biological methods to study

TMA and BSH compounds including

durability in serum lipophilicity receptor

affinities toxicity IC50 and apoptotic

pathway Two cells cultures were used

colon cancer cells HCT116 and healthy

colon cells CCD841 and two boron com-

pounds mercaptododecaborate dianion

trimethylammonium 1-hydroxy-1-carbado-

decaborate were used in this work [4]

Results

Due to their structure TMA and BSH are

easily coupled with proteins Additionally

by dint of showing poor EPR signal TMA

is detectable in biological structures Both

compounds crossed the cell membrane and

located in the cytoplasm Survivability of

the cells correlated with cytotoxicity of the

compounds tested The apoptosis pathway is

contingent on the concentration of the

compound tested not on the incubation

period


It is highly durable in solutions of diverse

pH and serum Consequently as a phar-

maceutical it could be administered orally

Moreover it is stable in all conditions tested

and has proper lipophilicity It does not

exclude the ability to cross the blood-brain

barrier The properties pointed out to create

an opportunity to enhance bioaccessibility

and extending of the half-life of the

compound

The project is financed from National

Science Centre (R No 201802XNZ7

03011)

References [1] WAG Sauerwein A Wittig et al Neutron

Capture Therapy Principles and Applications

2012 Springer-Verlag Heidelberg

[2] MA Gryziński M Maciak Appl Radiat Isot

vol10610-7 2015 DOI 101016japradiso2015

07047

[3] RL Moss Appl Radiat Isot vol 882-11 2014

DOI 101016japradiso201311109

[4] RF Barth P Mi et al Cancer Commun (Lond)

vol 1938(1)35 2018 DOI 101186s40880-018-

0299-7

(1)


Session 6 O 4

The structure of Nucleobindin-2 is regulated by divalent metal cations

Anna Skorupska1 Andrzej Ożyhar

1 Dominika Bystranowska

1

1Department of Biochemistry Molecular Biology and Biotechnology Wrocław University

of Science and Technology Wybrzeże Wyspiańskiego 27 50-370 Wrocław

Background

Nucleobindin-2 (Nucb2) is a multidomain

protein which possesses six domains signal

peptide IleLeu-rich region DNA-binding

domain two EF-hands domains acidic rich

region and leucine zipper motif [1] The EF-

hand domain is responsible for Ca2+ and

68

Mg2+ binding [2] Nucb2 exhibited a broad

range of expression in central nervous

system [3] and peripheral tissues [4] which

suggests that Nucb2 is involved in variety of

physiological processes The aim of this

study was to reveal the effect of Mg2+ and

Zn2+ binding on the structure of recom-

binant Nucb2 from red junglefowl (Gallus

gallus) Up to now CD spectroscopy and

limited proteolysis were applied The CD

analysis showed that only Zn2+ binding

resulted in alteration of the protein structure

This observation was consistent with limited

proteolysis data Only Zn2+ addition pro-

vided clear protection of Nucb2 against

proteolytical degradation Among divalent

metal cations Mg2+ and Zn2+ are the most

abundant ones in the human body The

modulation of the protein structure by

divalent metal cations may be significant for

both protein function and interactions


1 Purification Gallus gallus Nucb2 was

expressed in E coli cells Pure and non-

tagged Nucb2 was obtained in four steps

immobilized ion metal affinity chromato-

graphy (IMAC) His-Tag removal by

HRV3C protease second IMAC and gel

filtration The purity of the protein was

confirmed by SDS-PAGE and ESI-MS

2 Circular dichroism (CD) The far-UV CD

spectra were recorded with JASCO J-815

spectropolarimeter The measurements were

carried out for 10 microM Nucb2 in the absence

and presence of Mg2+ and Zn2+ at 20degC from

300 nm to 195 nm in triplicate The baseline

spectra of the buffer were subtracted

Secondary structure content was calculated

using CDNN software [5]

3 Limited proteolysis 10 microM ggNucb2 was

digested with endopeptidase Glu-C (V8)

(15000) in the absence (5 mM EDTA) and

presence of 10 mM Mg2+ and 100 microM Zn2+

The reaction was stopped at different time

intervals and analysed by SDS-PAGE The

digestion was also performed in broad range

of Mg2+ and Zn2+ concentration for 120 min

Results

The far-UV CD spectra recorded in the

absence and presence of Mg2+Zn2+ have

two negative maxima at 208 and 222 nm

which are characteristic for α-helical

structure [6] However the spectra deconvo-

lution showed that Nucb2 has a significant

amount of disordered regions too The Mg2+

exhibited no significant effect on the

secondary structure of Nucb2 However

Zn2+ increased the amount of α-helices and

decreased the content of unordered

structure Limited proteolysis results showed

that digestion patterns in the absence and

presence of Mg2+ are similar which

is in agreement with CD spectra results The

V8 digestion resulted in generation of two

fragments of 45 kDa and 30 kDa Inte-

restingly the presence of Zn2+ led

to limitation of accessibility of cleavage site

of Nucb2 which was caused probably by

the conformational change of Nucb2

molecule Presence of 100 microM Zn2+ led

to accumulation of fragments of 45 kDa

which seemed to be proteolytically resistant

Low Zn2+ concentration (0-10 M) has

a minor effect on the susceptibility to V8

digestion (similar to Mg2+)


In this paper we utilized CD and limited

proteolysis for initial characterisation of the

effect of divalent metal cations on Gallus

gallus Nucb2 and showed Zn2+ as the

specific ligand for the protein In particular

both secondary and tertiary structure of

Nucb2 are modulated by Zn2+ Interestingly

Mg2+ has not affected Nucb2 structure The

CD spectra showed that Nucb2 has

a significant amount of α-helical structure

and unordered regions The addition of Zn2+

led to an increase of the α-helical structure

and simultaneous decrease of the unordered

regions The molecule of Nucb2 in the

69

presence of Zn2+ undergoes structural

changes which probably leads to attain

a more compact structure The limited

proteolysis results also confirmed this

hypothesis Proteolysis occurres mainly

at the unordered regions rather than α-

helices [7] The addition of 100 microM Zn2+

provided a protection of Nucb2 against V8

digestion This Zn2+-dependent confor-

mational change of Nucb2 may have

implication in the physiological role of this

protein However the exact role of Nucb2-Zn2+

interaction required the further research

Acknowledgement

The work was supported by the NCN Grant

02NO001219

References [1] S Barnikol-Watanabe NA Gross et al Biol

Chem Hoppe Seyler vol 375 497-512 1994

101515bchm319943758497

[2] M Ikura Trends Biochem Sci vol 21 14-17

1996 101016S0968-0004(06)80021-6

[3] S Oh-I H Shimizu et al Nature vol 443 709-

12 2006 101038nature05162

[4] P Prinz A Stengel et al Curr Opin Pharmacol

vol 31 19-24 2016 101016jcoph201608011

[5] G Boumlhm R Muhr et al Protein Eng vol 5

191-195 1992 101093protein53191

[6] SM Kelly NC Price et al Biochim Biophys

Acta vol 1338 161-85 1997 101016s0167-

4838(96)00190-2

[7] A Fontana P Polverino de Laureto et al Fold

Des vol 2 17-26 1997 101016S1359-0278

(97)00010-2


Session 6 O 5

Stress affects the expression of lsquomajor housekeepingrsquo genes

ndash is phosphorylation involved

Aleksandra Tomczak12

Marta Pałka1 Jadwiga Jabłońska

1 Monika Woacutejcik

1

Ryszard Rzepecki1

1 Faculty of Biotechnology University of Wroclaw 2 Presenting and corresponding author

aleksandratomczakuwredupl

This work was funded by the Opus 11 grant from the National Science Centre Poland no

O-201621BNZ400541

Background

The process of transcription is one of the

key adaptive mechanisms and needs to be

strictly controlled in response to environ-

mental factors and stimuli (as heat shock)

Recently interest in this topic has been

growing among scientists because it is still

not known much about the mechanisms

controlling it

Heat shock is an invaluable model for

studying mechanisms regulating gene expres-

sion and is well known and easy to control

[1] Many papers report that during stress

transcription is shut down globally while

only a few loci are highly activated [2]

These active loci are connected with heat

shock proteins (Hsp) family which functions

as intra-cellular chaperones

Lamins are evolutionarily conserved proteins

classified as type V intermediate filaments

which are involved in the regulation of gene

expression chromatin organization DNA

replication and repair signaling develop-

mental regulation and nuclear positioning

[3] In order to play such a variety of func-

tions lamins interact with many different

nuclear proteins which are directly or

indirectly responsible for a particular func-

tion Lamins (the main component of the

nuclear envelope) together with associated

70

proteins built a complicated platform for the

regulation of nuclear processes It has been

proved that the chromatin regions located

near the nuclear envelope consist mainly

of heterochromatin ndash transcriptionally inac-

tive regions

Our research suggests that lamins and

associatedinteracting proteins are signi-

ficantly connected with transcription regu-

lation In this work we focus on changes in

gene expression profile in stress response

Our results also suggest that the phosphor-

rylation status of HSF and lamins changes

Does the phosphorylation cause the trans-

cription shut down or is it the result of it


Cell culture and heat shock treatment


D melanogaster embryonic cell lines ndash Kc

and S2 Cells were maintained in suspension

culture (in Schneider`s Drosophila Medium

from Gibco with 10 FBS and 1

antibiotic-antimycotic) at 23degC as normal

conditions To induce the heat shock cells

were incubated at 37degC for 1 h before

further experiments

Real-time quantitative PCR RNA-seq

and data analysis

Cells were lysed on plates and total RNA

was extracted For RT-qPCR ndash the cDNA

synthesis was performed RNA extractions

and cDNA synthesis from all samples were

performed for three biological replicates

RT-qPCR was performed using Quant-

Studiotrade 5 thermocycler and data were

calculated by connected Applied Bio-

systemstrade qPCR analysis module

For RNA-seq ndash mRNA enrichment library

construction and Illumina sequencing were

performed (Novogene) Raw data were pre-

processed mapped and analyzed using the

DESeq2 analysis pipeline in RStudio

Western blotImmunofluorescence and

analysis

Standard western blotimmunofluorescence

procedure was performed and data were

analyzed using Image LabImageJ software

Results

We have developed a protocol that allows

us to study the stress response in cells

We have found and functionally described

large changes in global transcription

in response to stress stimuli Our data show

that the level of some transcripts widely

considered as stable reference genes alter

after exposure to stress

RNA-seq analysis allowed us to select the

set of genes that remain stable in heat shock

response and between different cell lines

Kc and S2

We have shown that under stress the

phosphorylation of HSF and lamin Dm

occur We also have shown one of a stress-

dependent phosphorylation site in lamin Dm

ndash Ser25


Epigenetics is a field of the future So far

many chromatin remodelers with histones

and RNA polymerase II in front have been

identified as those in which post-trans-

lational modifications either activate or lead

to gene repression Global transcription shut

down is clearly visible in heat shock In this

study we show that working on models

such as stress response (and others causing

global expression alterations) scientists

should be very careful in choosing reference

genes for normalization ndash even with genes

widely considered as stable

In our project we show that during heat

shock specific phosphorylation of lamin

occurs on Ser25 which results in a change

in its solubility and potentially leads to

stronger binding of chromatin in stress

These data may indicate that lamins play

a key role in turning down gene

transcription

71

References [1] J Tower Exp Gerontol 46(5) 355-362 2011

DOI 101016jexger201009002

[2] JF Cardiello JA Goodrich et al Mol Cell Biol

2838(18) 2018 DOI 101128MCB00181-18

[3] M Pałka A Tomczak et al Cell Mol Biol Lett

23 32 2018 DOI 101186s11658-018-0093-1


Session 6 O 6

Male infertility in context of oxidative stress the analysis of Total

Antioxidant Status and clusterin concentration in human seminal

plasma ndash pilot study

Ewa Janiszewska1 Izabela Kokot

1 Iwona Gilowska

2 Ewa Maria Kratz

1

1Department of Laboratory Diagnostics Division of Laboratory Diagnostics Faculty

of Pharmacy Wroclaw Medical University Poland 2Laboratory of Infertility Diagnostics

Clinical Center of Gynecology Obstetrics and Neonatology Opole Poland

Background Infertility becomes a significant problem around the world especially in the deve-loped countries It is estimated that nearly 15 of couples of the reproductive age are trying to have a child without success In Poland this problem affects about million and a half of couples and the male factor alone contributes to about 50 of those cases [1 2] Despite the development of medical sciences there is lack of early sensitive male infertility biomarkers Semen analysis routinely performed during the male infertility diagnostics may result in no pathological values It concentrates mainly on the spermatozoa features Ninety-eight percent of human semen constitutes seminal plasma which is a mixture of many proteins playing a relevant role in the proper function of the male reproductive system and fertilization process Among them clusterin (CLU) is one of the important human seminal plasma glycoproteins playing crucial role in the proper sperm cells maturation as well as in the main-tenance of oxidative-antioxidative balance [3] Among many reasons of male inferti-lity an oxidative stress is considered as one of the most important [4] In this study we decided to determine clusterin concentration and assess the Total Antioxidant Status

(TAS) one of the oxidative-antioxidative balance parameters in seminal plasma of infertile men from astenozoospermic and teratozoospermic groups in comparison to the results obtained for normozoospermic patients

Material and Methods Seminal plasma samples of 72 infertile male partners of the reproductive age were collected in the Laboratory of Infertility Diagnostics Clinical Center of Gynecology Obstetrics and Neonatology Opole Poland The standard semen analysis was carried out according to WHO directives [2010] and based on its results patients were classified as teratozoospermic (n=26) astenoterato-zoospermic (n=19) and normozoospermic (n=27) The ejaculates were centrifuged (3500timesg 10 min RT) to obtain seminal plasma Seminal plasma clusterin concen-tration was determined using ELISA Kit (Human Clusterin Bioassay Technology Laboratory) The Total Antioxidant Status was estimated in patientsrsquo seminal plasma using automatic method (Randox Labora-

tories Ltd) in autoanalyser Konelab 20i Statistical analysis was performed using STATISTICA 133 PL (StatSoft Inc) software (U Mann-Whitney test)

72

Results The median value of clusterin concentration was visibly lower in the normozoospermic group in comparison to the teratozoo-spermic and astenoteratozoospermic patients The reversed trend was observed for median of TAS concentration However no statistically significant differences in the values of determined by us parameters between analysed seminal plasma groups were observed

Discussion and conclusions Seminal clusterin concentration in men with decreased fertilityinfertile seems to be an interesting factor when analysed in the context of oxidative-antioxidative balance

Further investigations on larger number of patients as well as the determinations of other oxidative stress parameters are needed in this field

References [1] S Baskaran A Agarwal A et al The World Journal of Menrsquos Health 2019 DOI 105534 wjmh180114 [2] M C Inhorn P Patrizio Human Reproduction Update 21(4) 411-426 DOI 101093humupd dmv016 [3] M Ferens-Sieczkowska B Kowalska et al Biomarkers 2013 18(1) 10-22 DOI 103109 1354750X2012719035 [4] D Milardi G Grande et al Fertility and Sterility 20129767-73 DOI 101016jfertnstert 201110013


Session 6 O 7

Volatile compounds as a means of protecting bacterial contamination

of cosmetics

Alicja K Surowiak Aneta Gwiazdowska Stanisław Lochyński Daniel J Strub 1Department of Chemical Biology and Bioimaging Wroclaw University of Science

and Technology 2Institute of Cosmetology Wroclaw College of Physiotherapy

Background

Microorganisms are omnipresent also

occupying our skin because of that

cosmetics are often contaminated Preser-

vation of everyday use products is important

field of cosmetic industry Moreover in

everyday use cosmetics are contaminated by

incorrect application All cosmetic products

should be tested on presence of pathogenic

and spoiling microorganisms Due to recent

trend in minimalizing chemical substances

in cosmetic formulation new antimicrobial

substances of natural source are sought

Furthermore possibility of combining

fragrance properties with antimicrobial

activity seams promising


Fifty-three volatile carbonyl compounds and

its oximes were tested Carbonyl com-

pounds were obtained from Merck Poland

except trans-cinnamaldehyde α-hexylcin-

namaldehyde p-tolualdehyde piperitone

that were obtained from Tokyo Chemical

Industry Co LTD piperonal that was

obtained from LOBA Chemie Austria and

vanillin that was obtained from Avantor

Performance Materials Poland SA All

corresponding oximes were synthetized in

our laboratory The correctness of oxime

structure was confirmed by GC-MS and

NMR and will be presented in further

studies All over hundred substances were

diluted in DMSO to the final concentration

of 30 mgmL Microorganisms used in this

study represent product spoiling species

commonly associated with beauty care

products Tested microorganisms were

obtained from Mecconti SARL Sp z oo

and consisted Gram negative Enterobacter

73

gergoviae Klebsiella aerogenes Burkhol-

dria cepacia Gram positive Kocuria

rhizophila Staphylococcus epidermis

Results

The best results was obtained by trans-

cinnamaldehyde citral trans-cinnamal

aldehyde oxime and saphranal oxime

against K aerogenes the MIC value was

300 microgmL Inhibitory activity of other test

agents was not satisfactory


Among all tested carbonyl compounds only

one ndash pseudoionone is excluded from use in

cosmetics Citral hexyl cinnamaldehyde

and α-isomethylionone are on list of subject

to the restrictions according to Regulation

(EC) No 12232009 of the European

Parliament and of the Council of 30

November 2009 on cosmetic products

Presented results shows that none of tested

substances are appropriate to be used as

preservatives in cosmetic formulation

exposed to tested microorganisms Although

those aroma compounds might enhance

activity of more familiar preservatives

Further test on other spoiling microorga-

nisms are necessary


Session 6 O 8

Studies on the reactivity of human serum IgG and IgA antibodies

with the bacterial OmpC protein as a potential diagnostic marker

of humoral immunodeficiency in children

Piotr Naporowski1 Danuta Witkowska

1 Aleksandra Lewandowicz-Uszyńska

2

Andrzej Gamian1

1Hirszfeld Institute of Immunology and Experimental Therapy Polish Academy of Sciences

Wrocław Poland 2Department and Clinic of Paediatrics Immunology and Rheumatology

of Developmental Age Wrocław Medical University Wrocław Poland

Background

OMPs (outer membrane proteins) are

immunologically important bacterial compo-

nents because they are mainly exposed on

the cell surface and may affect the physio-

logical functions of host tissue contributing

markedly to the mechanisms of patho-

genicity progression of infection and the

development of inflammatory response The

OmpC is a major protein in enterobacterial

outer membrane recognized by human

immune system Accessibility of OmpC to

host defense mechanisms make them

attractive as immunodiagnostic markers

potential immunoprophylactics and conve-

nient carriers for carbohydrate antigens

Preliminary research performed on mice

have showed that OmpC protein from

Shigella flexneri 3a plays a protective role

against enterobacterial infections [2] The

reactivity of OmpC with human sera

revealed low total levels of IgG and IgA

in immunodeficiency in children [1] The

peptide (RYDERY sequence) was identified

as an epitope recognized by antibodies

indicating the OmpC may serve as an

immunodiagnostic marker [2] Moreover

the peptide with OmpC epitope sequence

can be used for binding to carrier proteins

for better availability antigen for induction

of specific antibodies


Sera of patients have been obtained from

Medical University of Wrocław The OmpC

was isolated from Shigella flexneri 3a (PCM

1793) by valeric acid method and puri-

74

fication with gel filtration and ion exchange

chromatography BSA (bovine serum

albumin) conjugates with linearcyclic

peptide of epitope sequence from OmpC

protein was prepared as described [2] IgG

and IgA levels in sera of patients were

measured by ELISA assay [1-3]

Results

Degree of BSA substitution with peptides

was estimated by MALDI-TOF-MS The

OmpC protein and BSA-peptide conjugates

were analysed with SDS-PAGEimmuno

blotting assay to check its purity and immu-

noreactivity with human serum samples

The OmpC and peptide conjugates were

used in ELISA The IgG and IgA antibody

levels were determined in sera of immuno-

deficiency patients and in children with

recurrent respiratory tract inflammation

of which the level was compared to the

healthy controls


Results of ELISA assay show that the

reactivity of both IgA and IgG antibodies

with enterobacterial OmpC protein was

significantly lower in immunodeficiency

children than in healthy children and adult

blood donors and increased gradually with

age although values for IgA were more

distinct than those of IgG The hetero-

geneous correlations of specific total IgA

and IgG antibodies may come from the

various ages of child patients and different

deficits of specific antibodies depending on

the status of immunoglobulinopathy However

the results of this study are promising and

suggest that the OmpC from Shigella

flexneri 3a might serve as a specific

humoral immunodeficiency marker espe-

cially in IgA deficiency and recurrent

respiratory tract infections

References [1] D Witkowska E Masłowska et al FEMS

Immunol Med Microbiol vol 48 205-214 2006

DOI 101111j1574-695X200600137x

[2] A Jarząb D Witkowska et al PLoS One vol

8 e70539 2013 DOI 101371journalpone

0070539

[3] A Pawlowski G Kaumlllenius et al Vaccine vol

17 1474-1483 1999 DOI 101016S0264-

410X(98)00385-5

POSTER PRESENTATIONS

77

The molecular study of anticancer activity of novel synthetic

derivatives of naringenin

Dagmara Baczyńska1 Joanna Kozłowska

2 Mirosław Anioł

2 Jolanta Saczko

1

1Department of Molecular and Cellular Biology Faculty of Pharmacy with Division

of Laboratory Diagnostics Wroclaw Medical University Borowska 211A 50-556 Wrocław

Poland 2Department of Chemistry Faculty of Biotechnology and Food Science Wroclaw

University of Environmental and Life Sciences Norwida 25 50375 Wrocław Poland

Background

Flavonoids belong to a large group of natural

compounds found in many plants where

they play an important role in cell protect-

tion fruit coloring photosensitization and

plant growth regulation Numerous investi-

gations have confirmed their variety of

biological activities such as anticancer anti-

inflammatory antioxidant etc [12] Narin-

genin (4rsquo 5 7-trihydroxyflavone) is an

active form of naringin a substance com-

monly found in citrus fruits The biological

activity easy accessibility and low cost

of the extraction make naringenin an

attractive candidate for anticancer therapy

We have previously shown that novel

synthetic O-alkyl derivates of naringenin

and their oximes can act as effective

antitumor agents [3] The aim of this study

is to perform the molecular analysis of their

anticancer activity


The cytotoxic effects of examined com-

pounds on various cancer cell lines were

measured using sulforhodamine B assay

Phosphatidylserine exposure and membrane

integrity were investigated using RealTime-

GlowTM Annexin V Apoptosis and Necrosis

Assay (Promega) The activity of Caspase-3

and -7 was analyzed by Caspase-Glo37

Assay (Promega) The detection of procas-

pases and its active form was performed

using western blot method Nuclear DNA

was extracted from cells and analyzed by

electrophoresis to detect DNA fragmen-

tation Staurosporine was used as a reference

agent for the induction of apoptosis

Results

The results indicate that different type of

cancer cells are similarly sensitive to all

investigated compounds except for narin-

genin and staurosporine

The apoptosisnecrosis assay shows that

cytotoxic effect of alkyl derivatives of

naringenin is caused by the necrotic pathway

The substitution of the oxime group to these

compounds induces phosphatidylserine

exposure and cellular membrane disinter-

gration The results confirm that oxime 7-O-

decylnaringenin activates apoptosis in HT29

cells although time between the induction

of phosphatidylserine exposure and the loss

of membrane integrity was over 4 times

shorter than observed for staurosporine In

contrast oxime 74rsquo-di-O- butylnaringenin

simultaneously induces signals from both

annexin V and DNA dye This suggests that

the activated process differs from apoptosis

On the other hand following 74rsquodi-O-

butylnaringenin exposure we observed an

activation of the caspase pathway which

was manifested by the increased reactivity

of cleaved forms of caspase-3 and -7

Interestingly the increases of caspase -3 and

-7 activities are detected 4 hours after

treatment of the cells A similar effect is

observed for oxime 7-O-decylnaringenin

but the time of caspase activation is longer

than 12 hours The analysis of caspase 3

and-7 supports hypothesis that mechanism

78

of action of naringenin and its alkyl

derivatives differs from apoptosis

One of the last step of cell apoptosis is DNA

fragmentation To verify the hypothesis that

oxime derivatives can induce the apoptosis

process we performed an examination

of genomic DNA extracted from HT29

cells after 48-hours of treatment with

studied compounds The results prove that

only staurosporine can activate DNA

degradation in the cells


Our data show that despite the activation of

effector caspases 3 and 7 the mechanism of

action of oxime 74rsquo-di-O-butylnaringenin

and oxime 7-O-decylnaringenin differs from

typical apoptosis Further investigation

should be carried out in order to clarify

whether we have observed a new type of

cancer cell death or overlapping effects

of apoptosis and necrosis

This project was financed by the National

Science Centre of Poland Grant No

201621BNZ901904

References [1] Leonardi T Vanamala J et al Exp Biol Med

235 710-717 2010 doi101258ebm2010009359

[2] Tripoli E La Guardia et al Food Chem 104

466-479 2007 doi101016jfoodchem2006

11054

[3] Kozłowska J Grela E et al Molecules 24 679

2019 doi 103390molecules24040679


Changes in myosin light chains expression in the mechanism

of adaptation to oxidative stress

Marta Banaszkiewicz Anna Krzywonos-Zawadzka Agnieszka Olejnik Iwona Bil-Lula

Division of Clinical Chemistry and Laboratory Hematology Department of Medical

Laboratory Diagnostics Faculty of Pharmacy Wroclaw Medical University Borowska 211 A

Wroclaw Poland

Background

Myosin consists of two heavy chains

(MHC-α and MHC-β) and two pairs of light

chains essential (ALC1 ndash atrial and VLC1 ndash

ventricular) and regulatory (RLC) Oxidative

stress associated with heart diseases leads to

disruption of the balance between synthesis

and degradation of contractile proteins ndash it

has been shown to induce post-translational

modification of myosin light chains making

them more susceptible to degradation by

metalloproteinase 2 (MMP-2) This results

in the degradation of VLC1 in heart

ventricles and the increased expression of

ALC1 instead The aim of the study was to

investigate changes in the expression

of myosin light chains in rat cardiac

myocytes under the influence of increased

oxidative stress This study may be an

introduction to further research of cardiac

function and actin-myosin interaction as

a result of the replacement of VLC1 for

ALC1 during ischemiareperfusion (IR)

injury


Wistar rats hearts were perfused using the

Langendorff method The first group ndash IR ndash

underwent oxygen stabilization (25 min)

global ischemia (22 min) and oxygen

reperfusion (20 min) Aerobic control was

only introduced to aerobic conditions for 77

minutes Next atria were cut off from the

hearts and ventricles were taken for

analyses In 47th minute of the experiment

coronary effluents were collected from the

buffer flowing through the heart Lactate

dehydrogenase (LDH) activity was measured

in the coronary effluents to examine cardio-

79

myocyte damage after the ischemia RQ-

PCR was performed for ALC1 gene

Quantitative analysis of ALC1 VLC1 and

MMP-2 proteins was performed using

ELISA tests MMP-2 activity in heart

homogenates was assessed by gelatin

zymography

Results

LDH activity was significantly increased in

IR group in comparison to Aero group

(p=001) Expression level of ALC1 gene

was significantly higher in IR group

in relation to Aero group (p=0004) ALC1

protein content in hearts homogenates was

also significantly increased in IR group in

comparison to Aero group (p=003) VLC1

content in coronary effluents was substan-

tially increased in IR group in comparison

to Aero group (p=002) which confirmed

that VLC1 was released into extracellular

space MMP-2 concentration in heart homo-

genates was increased in IR group in

comparison to Aero group (p=003) The

activity of pro-MMP-2 and active-MMP-2

forms was also increased in IR group in

comparison to Aero group (p=004 p=003

respectively) Accordingly the total-MMP-

2 activity was higher in IR group in

comparison to Aero control (p=003)


In conclusion above data showed that

ischemia and reperfusion induced changes

in VLC1ALC1 already at the level of gene

expression There are many studies fasci-

nating in VLC1ALC1 replacement but the

researchers consider different heart diseases

for example progressive heart failure ven-

tricular aneurysmectomy familial hyper-

trophic cardiomyopathy [1 2] In general

they confirmed the existence of VLC1ALC1

replacement mechanism to improve heart

function [3 4] This preliminary study

provides the basis for further studies on

cardiac function changes actin ndash myosin

interactions and an influence of MMP-2 on

contractile proteins

This work was supported by the National

Science Centre [grant no UMO-201623

BNZ303151]

References [1] K Nakao H Yasue et al Circulation vol 86

1727-1737 1992 10116101CIR8661727

[2] J Machackova J Barta et al Canadian Journal

of Cardiology vol 22 953-968 2006 101016

S0828-282X(06)70315-4

[3] I Morano Journal of Molecular Medicine vol

77 544ndash555 1999 101007s001099900031

[4] GM Diffee EA Seversen et al American

Journal of Physiology-Heart and Circulatory

Physiology vol 284 H830-H837 2003 101152

ajpheart007612002


The unique content of oat oil ndash the perspectives of exploitation

Karol Banaś1 Joanna Harasym

12 Nathan Tancula

1 Agnieszka Orkusz

12 Remigiusz

Olędzki12

1Faculty of Production Engineering Wrocław University of Economics and Business

Poland 2Adaptive Food Systems Accelerator ndash Science Centre Wrocław University

of Economics and Business Poland

Background

In recent years there has been an increase in

consumer awareness of the benefits resul-

ting from consuming oat-based products [1]

The main advantage of oat products is their

good perception among consumers which

increase the willingness to buy Oats contain

many antioxidant compounds (polyphenols

avenantramids tocochromanol phytic acid

MUFA and PUFA including α-linolenic

acid melatonin inositol phosphates phyto-

80

sterols) and other biomolecules of well

recognized health impact (β-glucans) [2-4]

Compared to other major cereals like barley

wheat and rice oats are still underestimated

crop [5] Research continuously proves that

oats are very interesting plant which com-

position offers a variety of substances that

may in the future be used in the food pharma-

ceutical cosmetic and chemical industry [6-

8] Still unexploited oat oil contains a large

amount of polar lipids compared to other

oilseeds [9] In the future polar lipid

fractions may be widely used in the

stabilization of emulsions used in the food

industry eg for the production of chocolate

lubrication and baking pastes [10-11]


The oil contained in oats has not been used

much so far but the development of new

methods and techniques of lipid

fractionation allows to use its potential in

many industries Polar oat oil lipids such as

glycolipids and phospholipids contain

hydrophilic and lipophilic groups in their

structure

A change in the balance between these two

groups enables modification of emulsifying

properties of these lipids and affects the

stability of emulsions [12]

The most numerous phospholipids in oat oil

are phosphatidylcholine (PC) and

phosphatidyl ethanolamine (PE) [13]

digalactosyldiacylglycerol (DGDG) is the

glycolipid present in the highest amount

[14] In current dietary trends the

composition of food products becomes

a key factor for the consumer Such

approach forces industry into for instance

choosing the appropriate emulsifier which

then affects stability and then the product

quality [15] The need for food

diversification on the market is forcing the

search for a new solutions for food

preservation which also starts the demand

for emulsifiers with new properties The

answer could be the new emulsifiers from

polar lipids extracted from underestimated

raw materials like oats Due to the charac-

teristics of the process of obtaining oat oil

and low allergenicity of oat in total no allergic

risk is foreseen as is the case of emulsifier

of soy or chicken eggs origin [16]

References [1] Rasane P Jha A J Food Sci Technol 52 662-675 2013 httpsdoiorg101007s13197-013-

1072-1

[2] Butt Masood Sadiq Eur J Nutr 47 68-79 2008 httpsdoiorg101007s00394-008-0698-7

[3] Zieliński H Archemowicz B Zywn Nauk

Technol JakoscFood Sci Technol Qual 19 5-26 2012

[4] Harasym J Nauki inżynierskie i technologie3 57-70 2011

[5] Halima Ben N Khemakhem B J Oleo Sci

64 915-932 2015httpsdoiorg105650josess 15074

[6] Murphy EA Davis JM J Appl Physiol 97

955-959 httpsdoiorg101152japplphysiol 002522004

[7] Harasym J Study E Limited P 2016 1-7 [8] Gupta S Cox SBiochem Eng J 52 199-204

httpsdoiorg101016jbej201008008 2010

[9] Leonova S et al 2008 Analysis of oil composition in cultivars and wild species of oat

(Avena sp) Journal of Agricultural and Food

Chemistry 56(17) pp7983-7991 [10] Swedish Oat Fiber SWEOAT TM Oils

Information Sheet

[11] Swedish Oat Fiber AB Company website SweOat Available at httpwwwsweoatcom

[Accessed May 18 2017] [12] Friberg SE 1997 Emulsion stability In S E

Friberg amp K Larsson eds Food Emulsions New

York Marcel Dekker Inc pp 1-55 [13] Montealegre C et al 2012 Journal of

agricultural and food chemistry 60(44) pp10963-9

[14] Doehlert DC et al 2010 Polar lipids from oat kernels Cereal Chemistry 87(5) pp467-474

[15] McClements DJ 2016 Food Emulsions Principles Practices and Techniques 3rd ed CRC

Press Taylor amp Francis Group

[16]Younes M Aquilina G EFSA Journal 202018(1)5969 102903jefsa20205969 2019


81

Oat oil samples from polar and non-polar extraction characterisation

with near (NIR) and medium infrared (MIR) spectroscopy


12 Nathan Tancula

1 Remigiusz Olędzki

12

Agnieszka Orkusz12




Background


so far however the development of new

methods and techniques of lipid fractio-

nation creates new possibilities and expands

the use of particular oat oil components in

the food industry [1] cosmetic [2] and

pharmaceutical industry [3] The research

proves that oats are an interesting plant

containing in its rich composition a variety

of substances that may be used in the food

industry in the future eg in creating new

solutions for exclusion diets or functional

food products [4] The development of new


nation allowed to separate such components

from oat oil which have huge application

potential and have not been used before [5]


The oat oil was obtained by Soxhlet con-

tinuous extraction The extraction was

proceeded with two different solvents

ndash ethanol (998) and petroleum ether

(4060) and extraction time was 8h The

obtained oil samples were tested in near

(NIR) and medium infrared (MIR) The oil

spectra were measured on the Nicolet 6700

FT-IR spectrophotometer using the high-

performance diamond SMART iTX ATR

accessory and the NIR measurement

accessory

Results

Two different solvents used during extrac-

tion provided two distinct oil samples

Ethanol extracted oil sample was orangeish

while petroleum ether extraction resulted in

yellow-olive colour of the oat sample The

next easily observed difference was noted in

the consistency of obtained oil samples

Polar extraction sample (ethanol) was

notably thicker and maintained in room

temperature started to solidify while rising

the temperature caused liquefaction of

sample

Non-polar extraction with petroleum ether

provided liquid sample which only started

to solidify when stored in 4 [degC] Different

aroma was noted for oat oil samples being

pleasant bread-like for polar extraction

sample and oat aroma mixed with petroleum

scent for non-polar sample

The spectra obtained with different solvents

shows specific differences and especially at

3500 cm-1 the sample extracted with polar

solvent can be easily distinguished


The differences observed in the samples of

oat oil obtained are due to the difference in

polarity of the solvents used for extraction

and the associated different extraction

capacity The petroleum ether which is

non-polar in nature extracts mainly non-

polar lipids and the colouring matters

responsible for the greenish colour of the

sample Polar ethanol isolated the polar

fraction of lipids ie phospholipids and

glycolipids which caused the sample

to thicken at room temperature as opposed

to the sample obtained by extraction with

petroleum ether

82

Extraction with Soxhlets apparatus using

two different solvents allowed to isolate

different components from oats depending

on the molecular structure affecting their

polarity and properties

The Near Infrared (NIR) and Medium

Infrared (MIR) spectroscopy method allows

for a quick qualitative evaluation of oat oil

References [1] Erazo-Castrejoacuten S V Doehlert DC Cereal

Chem 78 243-248 2001

httpsdoiorg101094CCHEM2001783243

[2] wwwbiovelimpl date of access 01022020

[3] Moreau RA Doehlert DC Lipids 43 533-

548 2008 DOI 101007s11745-008-3181-6

[4] Harasym J Nauki inżynierskie i technologie

3 57-70 2011 httpswwwresearchgatenet

publication280088831_Obecny_status_owsa_

w_diecie_bezglutenowej_Present_status_of_oats_

in_the_gluten-free_diet

[5] Younes M Aquilina G EFSA Journal 2020

18(1)5969 2019 DOI 102903jefsa20205969


Myelin as an example of lyotropic liquid crystal

Dominika Benkowska-Biernacka Justyna Rojek Katarzyna Matczyszyn

Advanced Materials Engineering and Modelling Group Wroclaw University of Science

and Technology Poland

Background

Lyotropic liquid crystals can be observed in

complex biological systems for instance in

cell membrane and myelin sheath Both

consist of components with unique ability to

create bilayer structures ndash lipids They are

amphiphilic molecules which tend to self-

organize into lamellar structure in aqueous

conditions Myelin in contrast to most

biological membranes exhibits high ratio of

lipids to proteins This structure is a target

of various autoimmune diseases therefore it

is crucial to be able to analyse and visualise

fine changes of its arrangement [1] More-

over there are several possibilities to obtain

artificial myelin which can be use as model

of myelin sheath [2] Herein we present two

formation methods of myelin figures


In this study we used commercially available

12-dilauroyl-sn-glycero-3-phosphocholine

(DLPC) First to prepare myelin figures

a dry droplet of phospholipids was hydrated

Thus growth of myelin tubes could sponta-

neously occur on the edge of lipid plaque in

high magnification The elongated structures

were observed under the polarized light

microscope Additionally we performed an

experiment with a slowly evaporating droplet

Myelin growth was observed at the contact

line between the droplet and a barrier [3]

Results

Hydration of dry lipid plaque caused growth

of myelin figures As shown in Fig 1 they

are formed from the edge of the dry droplet

This material can be observed under crossed

polarizes which means that the tubes of

lipid bilayers exhibit birefringence Moreover

research which were performed with

a retardation plate showed that myelin tubes

are three dimensional structures In contrast

to results obtained by the first experiment

the second experiment on the system with

slow water evaporation gave smaller

diameter of myelin figures and was more

sensitive to temperature changes


Two different ways to obtain myelin tubes

are shown Each experiment allows us to

obtain different types of elongated myelin

structures Presumably quality and para-

meters of prepared material depend on

83

a factor which causes growth of tubes

Furthermore artificial myelin could poten-

tially be used as a model in biomedical

applications to mimic in vivo behaviour of

myelin sheath

References [1] Y Talmon R Bec J Am Chem Soc 138 37

12159-12165 2016 101021jacs6b04826

[2] L Tayebi M Mozafari et al International

Journal of Photoenergy 1-7 2012 1011552012

685617

[3] LN Zou Physical Review vol3 061502 2009

101103PhysRevE79061502

Fig1 Myelin figure under the polarized light microscope Scale bar 50nm


Preliminary view on Danio rerio and Homo sapiens otolin-1

Klaudia Bielak1 Anna Zoglowek

1 Piotr Dobryszycki

1

1 Department of Biochemistry Molecular Biology and Biotechnology Wroclaw University

of Science and Technology

Background

Mineralization of variable tissues in inver-

tebrates and vertebrates is a strictly regula-

ted process which involves action of many

distinctive intra- and extracellular proteins

[1] These proteins control the deposition of

inorganic material [2] Biomineral matrix

proteins are a wide variety group in which

intrinsically disordered proteins as well

as proteins structurally defined can be

distinguished [1] Here we present preli-

minary comparison of molecular properties

between recombinant Homo sapiens and

Danio rerio collagen-like otolin-1 [3]


Recombinant protein expression and

purification Danio rerio (dOtolin1) and

Homo sapiens (hOtolin1) otolin-1 was

expressed in Arctic Express Escherichia

coli in pQE80L plasmid for 24 h in 16oC

Protein purification included immobilized

metal affinity chromatography (IMAC) and

size exclusion chromatography (SEC)

84

Circular dichroism spectroscopy

Far UV CD spectra was recorded with Jasco

J-815 spectropolarimeter at 20oC between

260 and 200 nm with scanning speed of 20

nmmin every 05 nm with 3 accumulations

Nano-Differential Scanning Fluorimetry

Thermal shift assay was performed in

presence of variable calcium ions concen-

trations with Prometheus device (Nano-

Temper) The range of temperatures was

between 20oC and 110oC with the linear

increase of 5oCminute

Limited proteolysis

To analyse the susceptibility of specific

proteolysis of dOtolin1 and hOtolin1 the

time-limited proteolysis of both proteins

was performed in presence of V8 protease at

23oC in time periods between 10 and 210

minutes

Results


purification

The proposed way of recombinant dOtolin1

and hOtolin1 resulted in yields of 1 mg and

5 mg of protein per 1L of bacterial culture

respectively


The estimation of dOtolin1 and hOtolin1

secondary structure content by CDPro

shows differences in helical content bet-

ween them (in case of dOtolin1 10 higher

than in case of hOtolin1)


The presence of calcium ions increases the

thermal stability of both proteins hOtolin1

is more sensitive on calcium ions concen-

tration The first denaturation midpoint is

increased from 41oC (10mM EDTA) to

68oC in presence of 01mM Ca2+ In case of

dOtolin1 transition temperature is affected

by 1mM Ca2+ concentration and is equal to

867oC

Limited proteolysis

The outcome of the assay shows the

difference of the susceptibility of proteolytic

digestion with V8 protease of dOtolin1 and

hOtolin1 dOtolin1 is not affected by the

incubation with V8 protease for 35 hours

By contrast hOtolin1 first degradation pro-

ducts occur in 45 minutes after the start of

incubation


Danio rerio otolin-1 and Homo sapiens

otolin-1 are originated from two distantly

related species yet they fulfil homologous

functions providing the scaffold of fish

otolith and human otoconia The preliminary

analysis of dOtolin1 and hOtolin1 shows

differences in their molecular properties

The amount of helical content between short

collagen-like dOtolin1 and hOtolin1 differs

This difference can be an explanation of the

resistance of proteolytic lysis with V8

protease where dOtolin1 is less affected by

the action of the protease Additionally the

thermal stability of hOtolin1 is stabilized by

lower concentrations of calcium ions in

comparison to dOtolin1 In future the

analysis of the influence of post-transla-

tional modifications on the properties

of otolin-1 is planned

Acknowledgments


Science Center (Poland) [UMO-201519

BST1002148] and in a part by statutory

activity subsidy from the Polish Ministry of

Science and High Education for the Faculty

of Chemistry of Wroclaw University of

Science and Technology

References [1] R Hołubowicz et al Postępy Biochemii 61(4)

364-380 2015

[2] M Wojtas et al Advanced Topics in Biomi-

neralization 3-32 2012 DOI 10577231121

[3] E Murayama et al European Journal

of Biochemistry 2692 688-696 2002

85

Fig 1 The denaturation midpoint of dOtolin1 (A) and hOtolin1 (B) at a given EDTA and Ca2+

concentration presented as the first derivative of the ratio between intensity of fluorescence at 350 nm

and 330 nm in respect to temperature


Mitochondrial proteome changes by doxycycline may protect organ

graft against perfusion injury

Iwona Bil-Lula1 Anna Krzywonos-Zawadzka

1 Grzegorz Sawicki

2 Michael

Mosser3

1Department of Medical Laboratory Diagnostics Division of Clinical Chemistry and

Laboratory Hematology Wroclaw Medical University Wroclaw Poland 2Department

of Anatomy Physiology and Pharmacology University of Saskatchewan Saskatoon

Saskatchewan Canada 3Department of Surgery University of Saskatchewan Saskatoon

Saskatchewan Canada

Background

End stage renal disease is the final phase of

chronic kidney disease It is well known that

hemo- or peritoneal dialysis is life-saving in

patients who progress to chronic renal

failure [1] In North America approxi-

mately 75 of all solid organ transplants

performed are kidney transplants [2] The

transplantation organ from one person to

another is necessarily accompanied by

injury occurred during either warm or cold

ischemia

The preservation of kidneys for transplan-

tation relies mainly on hypothermia to

decrease cellular metabolism and conserve

stores of adenosine triphosphate Because

metabolism is ongoing albeit at a slower

rate the duration of cold ischemia should be

minimized as much as possible [3] Even

with machine cold perfusion significant

preservation injury nonetheless occurs and

likely contributes to delayed graft function

and acute tubular necrosis in the

transplanted kidney [4] Injury of any cause

is suspected to lead to a decrease in kidney

function shortened graft survival and an

increase in rejection due to increased

activation of the immune system [5]

It has been previously shown that doxy-

cycline (Doxy) protects the kidney from

preservation injury by inhibition of matrix

metalloproteinase However the precise

molecular mechanism involved in this

protection from injury is not known For this

reason the aim of the current study was to

86

assess the potential mitochondrial target for

doxycycline nephroprotection


Male Sprague-Dawley rats were used as a

surrogate model of ex vivo kidney perfusion

The left renal artery was ligated in situ for

10 minutes of warm ischemia then cannu-

lated and the kidney was removed and

rapidly cooled to 4degC The kidney was

perfused with a standard perfusion buffer

with the addition or without doxycycline

(100 microM) for 22 h Then tissue protein

extract from kidney and perfusates were

analysed by EM 2DE MS and biochemical

tests Graphpad Prism v 60 was used for

statistical analysis

Results

LDH NGAL and total protein levels were

measured in perfusates as the markers of

injury A significant increases in LDH activity

and NGAL levels were observed in per-

fusates from ischemic kidneys compared to

the controls 100 microM Doxy decreased cells

injury during cold perfusion (plt005) Elec-

tron microscopy confirmed that doxycy-

cline protected the kidney from the separa-

tion of cells and enlarging of the extra-

cellular space as well as from the formation

of dense bodies and mitochondria damage

(fig 1) Analysis of kidney homogenates by

2DE and identification by mass spectro-

metry revealed proteins such as N(G)N(G)-

dimethylarginine dimethylaminohydrolase

and phosphoglycerate kinase 1 were incre-

ased by Doxy in comparison to the controls


This study allowed to get the knowledge

about the specific mechanism by which

inhibition of MMPs protects kidneys from

cold preservation injury Data showed that

the maintenance of mitochondrial meta-

bolism and mitochondrial structure was the

main target of doxycycline nephroprotection

Study was supported by the National

Science Centre grant no UMO-2017

27BNZ400601

References [1] D Jain DB Haddad et al World J Nephrol

vol81-10 2019 doi 105527wjnv8i11

[2] Organ Transplant US Organ Donation System

[Internet] UNOS [cited 2019 Mar 4] Available

from httpsunosorgtransplant

[3] J Kox C Moers et al Transplantation

vol1021344-50 2018 doi 101097TP

0000000000002188

[4] D Bon N Chatauret et al Nat Rev Nephrol

vol8 339-47 2012 doi 101038nrneph201283

[5] JV Bonventre L Yang J Clin Invest

vol1214210-21 201 doi 101172JCI45161

Fig1 Representative electron micrographs of control rat kidneys (left micrographs) and perfused with

Doxy (right micrographs) 1500x magnification Doxy ndash doxycycline


87

In silico of study of imidazole based compounds as potent inhibitors

of p53-MDM2 interaction

Sebastian Błażkoacutew1 Kordian Rogalski

1 Żaneta Czyżnikowska

2

1Faculty of Pharmacy Wroclaw Medical University 50-556 Wrocław Poland 2Department

of Inorganic Chemistry Wroclaw Medical University 50-556 Wrocław Poland

e-mail zanetaczyznikowskaumedwrocpl

Background

It is widely known that p53 protein

influence many cell processes including

activation of DNA repair or induction of

apoptosis [1] The interaction of MDM2

with p53 is the most common reason of its

inhibition what disrupt many signaling

pathways The detailed mechanism of this

phenomenon is not fully understood

Structural findings proved that Leu26

Trp23 and Phe19 sequence in tumor

suppressor protein is the key factor Last

studies provide also suggestions that

inhibition of p53-MDM2 interaction leads

to activation of p53rsquos tumor suppressing

function [2] Therefore the p53-MDM2

interactions are extensively studied in terms

of anticancer agents design [1]


Computer-aided drug design become an

essential tool to discovery and analyze

compounds of the potential therapeutic

applications In the present project we used

computational approaches in order to design

of p53-MDM2 interaction inhibitors The

structures of proposed ligands were opti-

mized at the B3LYP6-31G level of

theory The molecular docking was performed

for compounds based on imidazole scaffold

and the human MDM2 protein originated

from the Protein Data Bank (PDB ID

3LBK) [3]

Results

Table The values of free energy of binding

and inhibition constant for most potent

compounds

Compound Free energy of

binding

[kJmol]

Inhibition

constant

A - 36 765 nM

B - 35 821 nM

C - 31 4 M

D - 31 4 M

E - 31 3 M

F - 35 843 nM

The most potent compound can bind to the

hydrophobic cavity of MDM2 and interact

similar to p53 (binding energy -36 kJmol)

Phe55 Leu57 Ile61 Tyr67 Phe91 and

Ile99 are the main amino acid residues

involved in hydrophobic interactions

Additionally the best inhibitor form

hydrogen bond with Leu54 (See Figure and

Table)


In the present study we predicted the nature

and strength of binding of imidazole

derivatives to MDM2 protein as a target In

accordance with the previous study the

proposed imidazole derivatives are able to

bind to the same binding cavity of protein

but shows better inhibition constants than

tested before[1]

Acknowledgments

Work was partially supported from

SUB008019029

88

References [1] S Shangary S Wang et al Annu Rev

Pharmacol Toxicol vol 49 223-241 2009

01146annurevpharmtox48113006094723

[2] A Twarda-Clapa S Krzanik et al J Med

Chem vol 60 4234-4244 2017

101021acsjmedchem7b00104

[3] GM Popowicz A Czarna et al Cell Cycle vol

9 1104-1111 2010 104161cc9610956

Figure Intermolecular interactions between compound A and human MDM2 protein

The hydrophobic forces are shown as arches and the hydrogen bonds as a line


The impact of xenoestrogens on the effectiveness of treatment

of hormone-dependent breast cancer

Kamila Boszkiewcz1 Ewa Sawicka

1 Agnieszka Piwowar

1

1 Department of Toxicology Faculty of Pharmacy Wroclaw Medical University Poland

Breast cancer is the most common cancer

among women and also causes the highest

number of cancer-related deaths in women

[1] In the treatment of hormone-dependent

breast cancers the most important is hor-

mone therapy including tamoxifen aroma-

tase inhibitors and their sequence [2] In the

pathogenesis of breast cancer xenoestro-

gens as an exogenous substances which can

interfere with the functioning of the endo-

crine system is the subject of many studies

Numerous publications confirm that many

compounds commonly found in the envi-

ronment can act as modulators of estrogen

receptors and thus compete or mimic the

action of endogenous estrogens (eg stimulate

the proliferation of cancer cells) This was

confirmed in in vitro and in vivo tests for ia

bisphenol A [3] and metalloestrogens [4]

Data on phytoestrogens are still unclear [5]

Definitely less is known about the impact of

xenoestrogens on the effectiveness of hor-

mone therapy used to treat breast cancer

and possible drug-xenoestrogen interactions

A systematic review of the literature derived

from PubMed Embase and Scopus rela-

ting to xenoestrogens in the context of

interactions with drugs used in breast cancer

hormone therapy was performed

89

Phytoestrogens in particular genistein are

the best studied xenoestrogen group This is

because phytoestrogens are often used to

control menopausal symptoms that occur in

patients who are receiving hormone therapy

Interaction between tamoxifen and genistein

in a postmenopausal breast cancer model

was demonstrated Adding low concen-

trations of genistein to tamoxifen causes a

reversal of its therapeutic effect (inhibition

of cell proliferation and arrest of the cell

cycle in the G1 phase) [6] Similar con-

clusions can be drawn from other studies

which showed that genistein at low doses

suppresses the therapeutic effect of tamo-

xifen Importantly high doses of genistein

do not suppress drug efficacy The majority

of supplements used to relieve menopausal

symptoms are rather multi-component

preparations containing several phyto-

estrogens whose effect may accumulate

The effects of both genistein and 8-pre-

nylnarygenin alone as well as four market-

based multi-component dietary supple-

ments on the effectiveness of 4-hydroxy-

tamoxifen and letrozole were studied Both

genistein 8-prenylnarygenin and all tested

supplements have been shown to activate an

estrogen receptor-dependent increase in

MCF-7 cell proliferation that has not been

inhibited by either 4-hydroxytamoxifen and

letrozole [7] Other studies revealed that

letrozole was shown to be effective in

inhibiting tumor growth in mice however

this effect was inhibited by the presence of

genistein [8] Examination of the effect of

xenoestrogens present in the diet (genistein

zearalenone) on the effectiveness of letro-

zole and palbociclib treatment using the

MCF-7 and T47D breast cancer cell lines

showed that the combination of letrozole

and palbociclib effectively inhibited the

proliferation of cancer cells while the

addition of both genistein and zearalenone

counteracted this effect [9] Bisphenol A

(BPA) is one of the best-tested for inter-

actions with drugs used to treat breast

cancer It has been shown that with the

simultaneous use of 4-hydroxytamoxifen

and bisphenol A the therapeutic effect of 4-

hydroxytamoxifen decreases This effect

was greater the higher the BPA concen-

tration [10] Another xenoestrogen ndash

methylparaben also contributes to the

occurrence of chemoresistance to drugs

used in the treatment of breast cancer

(tamoxifen fulvestrant) [11]

Due to widespread exposure to xenoestro-

gens as well as a steady increase in

incidence of breast cancer examining the

impact of endocrine active compounds on

the effectiveness of therapies used in the

treatment of hormone-dependent breast

cancer is becoming a clinically important

issue As shown in this literature review the

majority of research focused on phyto-

estrogens When analyzing the current state

of knowledge it seems that their intake

should be avoided during ongoing cancer

treatment An area requiring further research

is the analysis of the impact of xeno-

estrogens other than phytoestrogens eg

metalloestrogens on the effectiveness of

drugs used in the treatment of breast cancer

References

[1] World Health Organization Breast cancer

Available on the Internet httpswwwwhoint

cancerpreventiondiagnosis-screeningbreast-

canceren acces 02012020

[2] AGWaks et al Breast cancer treatment

A review JAMA 2019 321(3) 288-300

[3] ZWang et al Low-dose bisphenol A exposure

a seemingly instigating carcinogenic effect on

breast cancer Adv Sci 2017 4 1600248

[4] JLuevano et al A review of molecular events of

cadmium-induced carcinogenesis J Environ Pathol

Toxiol Oncol 2014 33 183-194

[5] CDuffy et al Implications of phytoestrogen

intake for breast cancer CA Cancer J Clin 2007

57 260-277

[6] JLJones et al Genistein inhibits tamoxifen

effects on cell proliferation and cell cycle arrest in

T47D breast cancer cells Am Surg 2002 Jun

68(6) 575-7

90

[7] M van Duursen et al Phytoestrogens in

menopausal supplements induce ER-dependent cell

proliferaton and overcome breast cancer treatment

in an in vitro breast cancer model Toxicology and

Applied Pharmacology 2013 269 132-140

[8] YHJu et al Dietary genistein negates the

inhibitory effect of letrozole on the growth of

aromatase-expressing estrogen-dependent human

breast cancer cells (MCF-7Ca) in vivo

Carcinogenesis vol 29 no 11 pp 2162-2168 2008

[9] BWarth et al Metabolomics reveals that dietary

xenoestrogens alter cellular metabolism induced by

palbociclibletrozole combination cancer therapy

Cell Chem Biol 2018 March 15 25(3) 291-300

[10] AH Goodson et al Activation of the mTOR

pathway by low levels of xenoestrogens in breast

epithelial cells from high-risk women Carcino-

genesis 2011 vol 32 no 11 pp1724-1733

[11] MALOsuna et al Methylparaben stimulates

tumor initiating cells in ER+ breast cancer models

J Appl Toxicol 2017 April 3(4)417-425


Antimicrobial activity of thyme tea tree and eucalyptus essential oils

against Staphylococcus aureus biofilm

Malwina Brożyna1 Justyna Paleczny

1 Karolina Dydak

1 Aneta Starzec

2 Adam Junka

1

1Department of Pharmaceutical Microbiology and Parasitology Wroclaw Medical

University 2Department of Pharmacognosy and Herbal Medicines Wroclaw Medical

University

Background

Microbial biofilm is responsible for plethora

of nosocomial infections [1] Moreover

increasing resistance of microorganisms to

antibiotics forces search for alternative

methods of infection treatment Essential

oils (EOs) which are compounds of high

antimicrobial potential are proposed as one

of the possible solution of aforementioned

issue [2 3]

The aim of the study was to determine the

activity of volatile and liquid phases of

selected essential oils against Staphylo-

coccus aureus bacterium


Three commercially-available essential

EOs eucalyptus (Eucalyptus globulus)

thyme (Thymus vulgaris) and tea tree

(Melaleuca alternifolia) were scrutinized

with regard to their activity against Staphy-

lococcus aureus ATCC 6538 To determine

Minimal Inhibitory Concentration (MIC)

and Minimal Biofilm Eradication Concen-

trations (MBEC) of aforementioned oils

serial dilution method was performed In

turn volatile phasersquos activity against

staphylococcal biofilm was assessed using

a self-developed test method

Results

Results revealed that MIC for eucalyptus

thyme and tea tree oil were 125 002

and 625 respectively while MBEC

values for these oils were gt50 018 and

25 respectively In case of analysis of

volatile fractions eucalyptus thyme and tea

tree oil reduced 60 80 and 70 of

staphylococcal biofilm respectively


EOs applied in this research display low

toxicity broad spectrum of effectiveness

biodegradability immune-stimulating and

anti-inflammatory properties [45]

Moreover the results obtained suggest that

application of essential oils against staphy-

lococcal biofilm may be considered effect-

tive approach Not only the liquid but also

the volatile phases of all EOs have shown

high efficacy against staphylococcal biofilm

The results concerning the activity of EOs

liquid phase are confirmed by numerous

91

scientific reports [6 7] while data on

activity of Eos volatile phase is still scanty

[8-10] Therefore this study presents another

step towards search of new treatment options

directed against staphylococcal biofilms

References [1] M S Blackledge R J Worthington et al Curr

ent Opinion in Pharmacology vol 13(5) 699-706

2013 doi101016jcoph201307004

[2] S Chouhan K Sharma et al Medicines vol

4(3) 58 2017 doi103390medicines4030058

C L Ventola Pharmacy and Therapeutics vol

40(4) 277-283 2015

[3] A K Pandey P Kumar et al Frontiers

in Microbiology vol 7 2161 2017

doi103389fmicb201602161

[4] N Mimica- Dukić B Bozin et al Planta

Medica vol 69(5) 413-419 2003 doi101055s-

2003-39704

[5] S D Cox C M Mann et al Journal of

Applied Microbiology vol 88(1) 170-175 200

doi101046j1365-2672200000943x

[6] K A Hammer CF Carson et al Journal

of Applied Microbiology 86(3) 446-452 1999

doi101046j1365-2672199900684x

[7] S Inouye T Takizawa et al Journal

of Antimicrobial Chemotherapy vol 47(5) 565-

573 2001 doi101093jac475565

[8] C Vasile M Sivertsvik et al Materials (Basel)

vol 10(1) 45 2017 doi103390ma10010045

[9] P Loacutepez C Saacutenchez et al Journal

of Agricultural and Food Chemistry vol 55(11)

4348-4356 2007 doi101021jf063295u


What is hidden in hop cones

Agnieszka Chwiłkowska1 Joanna Iwankiewicz

2 Hanna Baurska

2 Mirosław Anioł

3

1Department of Molecular and Cellular Biology Faculty of Pharmacy Wroclaw Medical

University Wrocław Poland 2Synkol Research amp Development Company Wroclaw

Poland 3 Department of Chemistry Faculty of Biotechnology and Food Science Wrocław

University of Environmental and Life Sciences Wrocław Poland

Background

Xanthohumol (XN) a prenylated chalco-

noid is a natural product found in the

female inflorescences of Humulus lupulus

also known as hops Besides XN hops

inflorescences also contain flavanones like

isoxanthohumol (IX) and 8-prenylnarin-

genin (8-PN) but at 10- to 100-fold lower

concentrations than XN respectively All

three substances are phytoestrogens they

naturally occur in plants and exert directly

or through metabolic changes estrogenic

effects because of structural similarity to

17β-estradiol [12] Aim of the paper was to

analyze the latest research applying 8PN as

a phytoestrogen


Data was collected by anlyzing available

articles which present results of studies

related to 8-PN XN and its association with

phytoestrogen Data were sought by com-

puter-based searches from databases

including PubMed Google Scholar Chosen

literature represent researches conducted

between 1999 and 2020

Results

Around 35-40 years of age women notice

a physiological decrease in the production

of their own female sex hormones Their

level which decreases with age initially

results in insignificant and then increasing

so-called traumatic symptoms of menopause

such as fatigue irritability problems with

concentration and memory sleep quality

deteriorates hot flashes dizziness and

headache trembling hands and occurring

palpitations Ailments can become a cause

of professional absence a decrease in pro-

ductivity and quality of work as well

as a deterioration in the quality of life for

women and their family members

92

The average diet of European women contains too little phytoestrogens to show their beneficial therapeutic effect Usually daily intake is 1-3 mg of phytoestrogens while women living on the Asian continent consume on average ten times more The high content of phytoestrogens in the diet of Asian women results in a decrease in their incidence in the perimenopausal period and the lack of osteoporosis after the menopause

Conclusions Supplementation of phytoestrogens in the perimenopausal and postmenopausal period seems to be the right choice due to health and socioeconomic benefits Hence the therapeutic treatment of 8-PN seems to be

very promising because it has been described as the most potent phytoestrogen found in nature [3]

References

[1] A K Żołnierczyk W K Mączka et al Fitoterapia vol 103 71-82 2015 DOI 101016jfitote201503007 Epub 2015 Mar 12 [2] K Štuliacutekovaacute M Karabiacuten et al Molecules vol 23 660-82 2015 DOI 103390molecules 23030660 [3] P Adegbola I Aderibigbe et al Am J Cardiovasc Dis vol 7 19-32 2017

The authors gratefully acknowledge funding from the National Science Centre (PL) under POIR grant number 010101-00-095618


Single treatment with Decitabine results in delayed morphological

changes

Kinga Chybicka12

Alicja Pawlak1 Ewa Zioło

1 Aleksandra Kaczorowska

3

Wojciech Kałas1

1Hirszfeld Institute of Immunology and Experimental Therapy Polish Academy of Sciences Wroclaw Poland 2Institute of Experimental Biology Faculty of Biological Sciences University of Wroclaw Poland 3Faculty of Fundamental Problems of Technology Wroclaw University of Science and Technology Wroclaw Poland

Background Decitabine (5-aza-2rsquo-deoxycytydine) is a hypomethylating agent approved for many haematologic malignancies treatment [1] It is also established that it can successfully sensitize colorectal cancer (CRC) cells for topoizomerase inhibitors in combination therapy [2] Even though the drug is in use for more than 40 years its mode of action is not fully examined yet Previous study showed that despite no direct cytotoxicity in colorectal cancer cells decitabine has more significant impact in prolonged culture During 1320 days of culture among observed changes were induction of many morphological abnormalities increase of p21 expression and reduction of cells clonogenity All of those may inform about

cell cycle inhibition connected to epithelial-mesenchymal transition or senescence

The aim of this study was quantitative measurement of morphological changes of CRC cells long-term culture after one-time decitabine treatment and study the mole-cular events underlying the change

Material and Methods HCT116 cells were incubated with 025μM decitabine for 5 7 and 14 days in standard culturing conditions Afterwards cells were fixed and stained for F-actin (ActinRed 555) and nucleus fluorescence (PureBlu Hoechst 33342) as well as SA-β-galactosidase acti-vity (CellEvent Senescence Green) Positive control for senescence were HCT116 cells incubated with 50nm Doxorubcin for 72h Results were obtained by fluorescence

93

microscopy and flowing cytometry then processed with Flowing Software Leica LasX and ImageJ

Results

Microscopic images analysis presented

bigger average nucleus area after decitabine

exposure Cytofluorimetric fluorescence

intensity measurement of HCT116 cells

showed higher F-actin expression even 7

days after treatment and indicated SA-β-

galactosidase activity Observed changes

were time-dependent and correlating with

worsening cells condition


Those results show that induced by

decitabine morphological changes were

reflected by increased F-actin quantity and

bigger nucleus area which is likely

a manifestation of drug-induced senescence

Acknowledgements

This research was supported with grant

OPUS 201725BNZ502608 and statute

programme IITD 32019

References [1] Hackanson B et al Recent Results Cancer Res

201269-97 2014 doi 101007978-3-642-54490-

3_18

[2] Pawlak A et al BMC Cancer 16(1)893 2016

doi 101186s12885-016-2925-6


Influence of manufacturing parameters on alginate-gelatin hydrogels

for 3D cell culture

Karolina Cierluk1 Agnieszka M Jankowska

2 Magdalena B Łabowska

2

1Faculty of Chemistry Wrocław University of Science and Technology 2Mechanical

Faculty Department of Mechanics Materials and Biomedical Engineering Wrocław

University of Science and Technology

Background

Hydrogels as cross-linked polymeric net-

works by the content of hydrophilic groups

are materials able to bind large amounts of

water They have been present in medical

applications since the 1960s and nowadays

are suitable for pharmaceutical applications

[1] Hydrogel in vitro cell culture media are

relevant to sustain cells and create an en-

vironment similar to the in vivo conditions

due to their high permeability to oxygen

nutrients or other water-soluble compounds

furthermore they allow cells to migrate

freely in any direction For this purpose

either natural (eg alginate chitosan

collagen fibrin gelatin) and synthetic poly-

mers are used Natural polymers are widely

used for their similarity of mechanical

properties to human tissues and their

biodegradability Unfortunately their dura-

bility is limited and their composition may

be variable [2 3]

There are technologies available for culturing

cells in 3D such as hanging drop method

low-binding plastic pyramid plates rotary

cell culture scaffold based cultures etc

Technologies of alginate hydrogels include

beads delayed gelation systems macro-

porous scaffolds honeycomb scaffolds with

pore structure scaffolds with nanoparticles

and 3D printed scaffolds Stem cell diffe-

rentiation and alginate hydrogels elasticity

matching to most types of tissue can be

controlled by optimizing type of alginate its

concentration and selection of cross-linking

technology Bioprinting may use materials

(eg alginate hydrogel as a bioink) and

cells they should be biocompatible to form

a variety of 3D formats where cell function

and viability are preserved within the

printed structure [3 4]

94

The aim of present paper is evaluation of

parameters manufacturing and material

influence on maintenance culture of tumor

cells from the line MCF-7DOX breast

cancer cells and describe fabrication of

alginate-gelatin hydrogel media for the cell

culture as well as assessment of mechanical

properties of cross-linked hydrogel Algi-

nates are naturally occurring anionic poly-

mers obtained from brown seaweed able to

form a gel in the presence of bivalent ions

eg Ca2+ They are used often due to their

rheological properties biocompatibility as

well as lack of toxicity Sodium citrate che-

lates calcium ions and is used to dissolved

cross-linked gels Changed hydrogel by

sodium citrate can be more suitable envi-

ronment for cells ndash can be printed and retain

their capacity to proliferate and group [1 5]


In this paper a line of MCF-7DOX tumour

cells has been used for the investigation

Hydrogel cell culture media were created

using physiological buffered saline (PBS)

with 5 alginate and 20 gelatine com-

position The hydrogel cross-linking was

carried out chemically with calcium ions

in CaCl2 solutions Sodium citrate was added

for controlled dissolved alginate gel [5]

Cell formation was observed on different

configurations of alginate hydrogel substra-

tes depending on the presence of sodium

citrate as well as the structure of cross-

linked hydrogel created by the appropriate

amount of calcium ions

Results

In this study has been achieved optimal

protocol to obtain alginate-gelatin hydrogel

which is sterile and non-toxic for cell

culture It has been also conducted evalu-

ation of sodium citrate on viability of MCF-

7DOX cells


In this work has been realized the initial

optimization of alginate-gelatin hydrogel

obtaining as a bioink for 3D printing Cross-

linking of the hydrogel has an effect

on obtaining desired mechanical properties

by selection of appropriate CaCl2 solution

concentrations which in consequence

influences the cell culture growth

References [1] A Kaczmarek-Pawelska Alginate-Based

Hydrogels in Regenerative Medicine 2019 DOI

105772intechopen88258

[2] A Stefanek T Ciach Biomimetyczne matryce

do przestrzennej hodowli komoacuterek Inż Ap Chem

vol 53(4) 294-295 2014

[3] M Topuz B Dikici et al A Review on the

hydrogels used on 3D bio-printing International

Journal of 3D Printing Technologies and Digital

Industry vol 2(2) 68-75 2018

[4] T Andersen P Auk-Emblem et al 3D Cell

Culture in Alginate Hydrogels Microarrays (Basel)

vol 4(2) 133-61 2015 DOI 103390

microarrays4020133

[5] Z Wu X Su et al Bioprinting three-

dimensional cell-laden tissue constructs with

controllable degradation Scientific Reports

624474 2016 DOI 101038srep24474


Metabolic differences between subcutaneous and visceral adipose

tissues based on gene expression study

Aneta Cierzniak1 Krzysztof Kaliszewski

2 Małgorzata Małodobra-Mazur

1

1Medical University in Wroclaw Department of Forensic Medicine Molecular Techniques

Unit Wroclaw 2Department of General Minimally Invasive and Endocrine Surgery

Wroclaw Medical University Wroclaw

95

Background

Adipose tissue is the main energy reservoir

in the body Besides the energy storage it

plays the role of endocrine organ secreting

numerous biologically active peptides called

adipokines Pathological and excessive

accumulation of adipose tissue both vis-

ceral (VAT) and subcutaneous (SAT) is

influenced by metabolic psychological

endocrine and genetic factors [1] Many

scientific reports indicate a high correlation

between obesity and the aberrant gene

expression profile [2] Although a greater

impact on the development of metabolic

disorders is attributed to VAT it turns out

that SAT may play an equally important

role in this process [3] Therefore we have

attempted to compare the metabolism

activity of both types of adipose tissue For

this purpose we have analyzed in VAT and

SAT the group of 27 genes associated with

the insulin pathway adipokines cytokines

lipids and transcription factors regulating

the development and metabolism of adipo-

cytes and transcription factors regulating

cell responses to hypoxia


Visceral and subcutaneous adipose tissue

biopsies were collected during abdominal

surgeries from 18 patients in the BMI range

from 20 to 27 in the age from 40 to 60

years with HOMA-IR lt 25 and with equal

distribution of sex The total RNA was

isolated using a combination of trizol method

and commercial column spin method kits

Reverse transcription was performed with

the use of High Capacity cDNA Reverse

Transcription Kit Gene expression was

done using Real-Time PCR based on SYBR

Green assay A relative gene expression

level normalized to the housekeeping gene

(β-actin) was calculated using the delta-

delta Ct (ΔΔCt) model

Results

A comparative analysis showed strong

statistical significant differences in the

expression between VAT and SAT occur in

the case of LEP (Leptin) (p = 0022) and

IGF2 (Insulin like growth factor 2)

(p = 0002) It was observed that the

expression of LEP was about twice lower in

VAT than in SAT but in case of IGF2 was

about twice higher in VAT than in SAT

Moreover in visceral adipose tissue we

observed also significantly increased

expression level of Il-10 (Interleukin 10)

PIK3R1 (Phosphoinositide-3-kinase regula-

tory subunit 1) CEBPβ (Enhancer binding

protein beta) TNFα (Tumour necrosis

factor alpha-like) and PPARGC1A (PPARG

coactivator 1 alpha) and significantly

decreased expression level of SLC2A4

(Solute carrier family 2 member 4) SCD1

(Stearoyl-CoA desaturase) and Il-6 (Inter-

leukin 6) compare to subcutaneous adipose

tissue but these results didnrsquot show

a statistical significance


The obtained results indicate metabolic

differences between VAT and SAT Sub-

cutaneous adipose tissue is seems to be

much more involved in the process of

lipogenesis which is indicated by increased

expression of genes associated with lipid

metabolism especially in fatty acids syn-

thesis (SCD1) and also by increased expres-

sion of gene encoding a leptin which is

considered an energy sensor that regulates

appetite and the amount of adipose tissue in

the body While the higher expression level

of LEP is probably the result of the bodys

response to increased lipid production In

turn visceral adipose tissue shows increased

susceptibility to inflammation which is

indicated by increased expression of genes

encoding inflammatory factors However

the reduced expression of SLC2A4 in VAT

with the simultaneously significant incre-

ased expression of IGF2 and also higher

96

expression of PIK3R1 compere to SAT may

indicate a greater risk of disturbances in the

insulin pathway which may lead to the

development of insulin resistance in

adipocytes in this type of adipose tissue

Financial support

The study is supported by The National

Science Centre (number of the research

project 201621DNZ500155) The research

protocols and all procedures were approved

by the Ethical Review Board of Wroclaw

Medical University Approval No KB-

1242017

References [1] D M Nguyen H B El-Serag The Epide-

miology of Obesity Gastroenterology clinics of

North America vol 39 2010 doi 101016jgtc

200912014

[2] SB Sonne R Yadav Obesity is associated with

depot-specific alterations in adipocyte DNA

methylation and gene expression Adipocyte vol 6

124-133 2017 doi 101080216239452017

1320002

[3] M D Barberio E P Nadler et al Comparison

of visceral adipose tissue DNA methylation and

gene expression profiles in female adolescents with

obesity Diabetol Metab Syndr vol 11 2019 doi

101186s13098-019-0494-y


Long-term treatment with indomethacin increases the number

of PACAP-immunoreactive porcine duodenal neurons

Marta Czajkowska1 Barbara Jana

2 Jarosław Całka

1

1Department of Clinical Physiology Faculty of Veterinary Medicine University of Warmia

and Mazury Olsztyn 2Division of Reproductive Biology Institute of Animal Reproduction

and Food Research Polish Academy of Sciences Olsztyn

Background

Due to numerous therapeutic applications

and high availability non-steroidal anti-

inflammatory drugs (NSAIDs) are the most

widely used drugs worldwide [1] Enteric

neurons are characterized by considerable

chemical plasticity and the appearance

of a pathological factor results in a change

in the synthesis of neurotransmitters [23]

Therefore the aim of the study was to

determine the effect of inflammation caused

by indomethacin supplementation on pituitary

adenylate cyclase-activating peptide (PACAP)

expression in enteric duodenal neurons in

domestic pigs


The study was carried out on eight imam-

ture pigs of the Pietrain x Duroc race

(approximately 20 kg of body weight about

8 weeks old) The animals were divided into

two groups ndash a control (C group) and

an experimental group (I group) Group C

(n=4) was consisted of animals which

received empty gelatine capsules Group I

(n=4) was composed of pigs which

indomethacin (10 mgkg b w) were given

orally for 4 weeks approximately 1 h before

feeding After this time animals from both

groups were euthanized Then frozen

sections (14 μm thickness) were prepared

from the collected material (3 cm fragments

of duodenum located 10 cm caudal to

musculus sphincter pylori) and subjected to

double immunofluorescence staining Anti-

bodies against the neuronal marker PGP 95

and against the pituitary adenylate cyclase-

activating peptide were used as primary

antibodies The secondary antibodies

ndash Alexa Fluor 488 and 546 ndash were also used

for staining Analysis of the sections was

performed using an Olympus BX51

fluorescence microscope

97

Results

Microscopic analysis showed significant

increase in the number of PACAP positive

neurons both in the myenteric and sub-

mucous plexuses of the porcine duodenum


Increased number of the PACAP-immuno-

reactive neurons in the myenteric and

submucous plexuses following indomethacin

evoked duodenal inflammation may reflect

down regulation of the inflammatory process

The results show that indomethacin through

inhibition of cyclooxygenase and thus

prostaglandins synthesis impairs the mucus

bicarbonate duodenal barrier To restore

intestinal homeostasis and counteract inflam-

mation local enteric neurons are subject to a

chemical adaptation process To synthesis

and release an additional volume of protective

neurotransmitters such as PACAP the ENS

recruits additional neurons thus increasing

the number of operating cells [2] Since the

chemical plasticity of the enteric neurons

constitutes the basis of gastrointestinal

compensatory mechanisms the presented

results may contribute to the future deve-

lopment of new strategies for the treatment

of gastrointestinal diseases

This study was supported by the National

Science Centre (grant no 201829

NNZ400348)

Tab 1 Percentages of immunoreactive enteric

neurons in the porcine duodenum in the control

and indomethacin-treated animals p lt 001

Active

neuronal

substance

Type of

plexus

Control

group

Indomethacin

group

PACAP

MP 969 plusmn

037

1191 plusmn 013

OSP 1051 plusmn

031

1402 plusmn 021

ISP 1282 plusmn

035

1636 plusmn 026

p lt 0001 indicate differences in PACAP

expression in comparisons to the control

animals

References [1] Adebayo D Bjarnason I Is non‐steroidal

anti‐inflammaory drug (NSAID) enteropathy

clinically more important than NSAID gastropathy

Postgrad Med J 2006 82(965) 186ndash191 DOI

101136pgmj2005039586

[2] Czajkowska M Rychlik A et al Long-term

treatment with naproxen changes the chemical

coding of the porcine intramural duodenum

neurons Ann Anat 2019 Oct 11227151425 DOI

101016jaanat2019151425

[3] Yagi K Takehara K et al Effects of pituitary

adenylate cyclase activating polypeptide-27 on

alkaline secretory and mucosal ulcerogenic

responses in rat duodenum Life Sci199863

(5)317-25 DOI 101016S0024-3205(98)00280-X


The relationship between TNF-α CDKN1Ap21 and MMP9

in esophageal squamous cell carcinoma

Maja Dorociak Katarzyna Augoff

Department of Surgical Education Wroclaw Medical University

Background

Esophageal squamous cell carcinoma

(ESCC) a highly aggressive and often late

diagnosed disease is mostly induced by

chronic inflammation [1] Inflammation

generally plays crucial role in tumorige-

nesis tending to promote cancer invasion

and metastasis the latter being clinically the

most critical aspect of the disease [2] Thus

most elements of the machinery involved in

inflammation-driven cancer progression

may work as a target for therapy In the

98

multistep process of metastasis remodelling

of extracellular matrix (ECM) allows cancer

cells to invade and migrate through their

microenvironment Matrix metallopro-

teinases (MMPs) are crucial for the ECM

degradation and they determine the aggressi-

veness of malignant cells Gelatinase MMP-

9 was found to be commonly upregulated in

human cancers and besides cleaving type

IV collagen laminin and elastin it affects

cell signalling by processing chemokines

growth factors or cell receptors [3 4] This

enzyme is also a major gene controlled by

transcription factor NFκB which states the

link between metastasis and TNF-α an

inflammation cytokine and mentioned

signalling pathway activator Recently it

was shown that cyclin-dependent kinase

inhibitor (CDKN1Ap21) plays an impor-

tant regulatory role in TNF-α-induced

MMP9 gene expression in triple-negative

breast cancer [5] CDKN1Ap21 apart from

arresting cell cycle can protect the cell from

apoptosis yet its actual role as either tumour

suppressor or an oncogenic factor seems

to be environment-dependent


In this study using real-time PCR and

immunohistochemistry (IHC) we investi-

gated levels of CDKN1Ap21 MMP9 and

TNF-α expression on both mRNA and

protein levels in ESCC tissues and the

relationship between CDKN1Ap21 and

MMP9 in human squamous cancer cells of

the esophagus KYSE70 by transfecting

cells with CDKN1Ap21 siRNA and treating

them with TNF-α

Results

Using real-time PCR we found that the

expression of CDKN1Ap21 and MMP9 as

well as TNF-α genes was significantly incre-

ased in cancer tissues compared to the control

groups These results were confirmed by

IHC We also found that the TNF-α treatment

of human esophageal squamous cancer cells

in in vitro conditions resulted in the

statistically significant increase in expression

levels of both CDKN1Ap21 and MMP9

Next using gelatin zymography we

observed that siRNA-induced transcriptional

silencing of CDKN1Ap21 gene inhibited

TNF-α-dependent MMP9 expression

Conclusions

CDKN1Ap21 may play an important role

in the development of ESCCs by its

contribution to the regulation of TNF-α-

induced MMP9 expression

References [1] Napier K J Scheerer M et al World Journal

of Gastrointestinal Oncology vol 6(5) 112-120

2014 DOI104251wjgov6i5112

[2] Balkwill A Cancer Metastasis Rev vol 25

409-416 2006 DOI101007s10555-006-9005-3

[3] Augoff K Hryniewicz-Jankowska A et al

Oncology Reports vol 31 2820-2826 2014

DOI103892or2014316

[4] Egeblad M Werb Z Nature Reviews vol 2

161-174 2002 DOI 101038nrc745

[5] Zaremba-Czogalla M Augoff K et al

Cellular Signalling vol 47 27-36 2018 DOI

101016jcellsig201803010


Singlet oxygen photogeneration by biological staining dyes

Alicja Duda1 Bartosz Kopyciński

2 Agata Blacha-Grzechnik

1

1Department of Physical Chemistry and Technology of Polymers Faculty of Chemistry

Silesian University of Technology Gliwice Poland 2Department of Engineering Materials

and Biomaterials Faculty of Mechanical Engineering Silesian University of Technology

Gliwice Poland

99

Background Photodynamic therapy synthesis of fine chemicals or wastewater treatment these are few of the most important uses of singlet oxygen (1O2) photogeneration [1] The signi-ficance of singlet oxygen in chemistry biology or medicine is highly appreciated by researchers around the world It is often formed in photosensitization processes where photoactive compound and source of light of appropriate wavelength are required [2] Considering great photoactive properties biological dyes have variety of applications thus they can be used as a photosensitizers In this work compounds such as tropaeolin rose bengal and crystal violet were tested for their photoactive properties

Material and Methods Tropaeolin rose bengal and crystal violet solutions in methanol (Sigma Aldrich) were used in singlet oxygen photogeneration Measurements were done using UV-Vis spectroscopy in quartz cuvettes by investi-gating homogeneous mixtures of given compound with specific chemical trap ndash 13-diphenylisobenzofurane (DPBF Sigma Aldrich) Source of light was lasers working at wavelength 532 nm

Results UV-Vis measurements were based on tracking in decrease absorbance at wave-length appropriate for chemical trap (410 nm) Total decreases of absorbance stand at 059 106 and 097 for tropaeolin (from

107 to 048) rose bengal (from 115 to 009) and crystal violet (from 111 to 014) respectively Measurements total time in each case was 240 seconds

Discussion and conclusions Photogeneration of 1O2 occurs when light-activated molecules of photosensitizer are causing excitation of oxygen molecule from its ground state Chemical traps such as DPBF are used to indicate this process by observing decrease of absorbance men-tioned in results That indeed is an outcome of the DPBF reaction with oxygen mole-cules which results in its oxidation

All given compounds have shown relatively good ability to singlet oxygen generation in homogeneous systems This gives opening to preparation of heterogeneous 1O2 photo-generation systems in which photo-sensitizers can be deposited on solid surfaces

This work was supported by European Social Fund in the framework of the project Silesian University of Technology as a Center of Modern Education based on research and innovationrdquo POWR030500-00-Z09817

References [1] M C DeRosa RJ Crutchley Coordination Chemistry Reviews vol 233-234 351-371 2002 DOI101016S0010-8545(02)00034-6 [2] S Takizawa R Aboshi et al Photochemical amp Photobiological Sciences vol 10 895-903 2011


Long-term melatonin treatment accelerates myocardial activation

processes

Aleksandra V Durkina1 Olesya G Bernikova

1 Ksenia A Sedova

2 Jan E Azarov

1 3

1 Department of Cardiac Physiology Institute of Physiology Federal Research Centre Komi Science Center Ural Branch of Russian Academy of Sciences Syktyvkar Russia 2Department of Biomedical Technology Faculty of Biomedical Engineering Czech Technical University in Prague Kladno Czech Republic 3Department of Biochemistry and Physiology Institute of Medicine Pitirim Sorokin Syktyvkar State University Syktyvkar Russia

100

Background

Melatonin is thought to have antiar-

rhythmic properties in ischemiareperfusion

conditions Previous studies from our group

demonstrated that the antiarrhythmic effect

of melatonin was associated with improved

ventricular activation but exact mechanisms

are unclear The improvement of activation

may be caused by enhancement of propa-

gation via His-Purkinje system andor

intramyocardial conduction The present

study aimed to assess effects of long-term

melatonin treatment on epicardial activation

time (AT) and conduction velocity (CV) in

rat hearts


Experiments were performed in a total of 44

anesthetized open-chest male Wistar rats

The animals received melatonin (10 mgkg

day single oral dose) or placebo for seven

days Unipolar electrograms were recorded

from the epicardium of the right ventricle

(RV) and left ventricle (LV) using an array

of 64-leads In each lead AT was deter-

mined as an instant of dVdt min during

QRS complex and isochronic activation

maps were constructed CV was measured

during electrical stimulation (400 bpm

2 mA 2 ms) in the middle of the LV and

RV free walls Anisotropy of conduction

was estimated as a ratio between a longi-

tudinal (CV max) and transversal (CV min)

CVs

Results

Melatonin reduced ATs in the LV

(1114plusmn096 vs 131plusmn071 ms plt0001) and

RV (108plusmn078 vs 1171plusmn099 ms p=0010)

in the melatonin (n=13) and control (n=12)

groups respectively In the LV CV

demonstrated marked anisotropy CV max

(074plusmn017 vs 073plusmn013 ms) CV min

(043plusmn012 ms vs 036plusmn010 ms) and

CV maxCV min (18plusmn05 vs 21plusmn07) did

not differ significantly in the melatonin-

treated (n=10) and untreated (n=8) animals

respectively However the RV CV being

isotropic was higher in the melatonin-

treated animals as compared to controls

(066plusmn01 vs 049plusmn01 ms p=0027

respectively)


Long-term melatonin treatment led to

myocardial activation enhancement which

was at least partly due intramyocardial

conduction acceleration in the RV On the

other hand activation time shortening by

melatonin in the LV implies involvement of

conduction system

The study was supported by Russian Science

Foundation (RSF 18-15-00309)


The antimicrobial effectiveness of bacterial cellulose dressings

chemisorbed with commonly used wounds irrigation agents against

chosen opportunistic pathogens

Dydak Karolina1 Paleczny Justyna

1 Brożyna Malwina

1 Junka Adam

1

Bartoszewicz Marzenna1

1Department of Pharmaceutical Microbiology and Parasitology Faculty of Pharmacy

Wroclaw Medical University Wroclaw Poland

Background

Bacterial bionanocellulose (BC) is a bioma-

terial produced by bacteria Komagataei-

bacter xylinus BC has a number of pro-

perties which make it an excellent bioma-

terial for medical applications The previous

research indicated that BC facilitates the

101

proper healing process by the maintenance

of optimal hydration of wounds The high

water-related properties of bacterial cellu-

lose allows to enrich the dressing with

various substances including antimicrobial

agents [12] Topical antibiotic therapy is

not recommended to treat wound infections

The reason is poor penetration of antibiotics

into the wound and a high risk of selecting

antibiotic-resistant strains For wound irri-

gation sterile antiseptics or lavaseptics are

recommended The octenidine dihydro-

chloride (OCT) polyhexamethylene bigu-

anide (PHMB) and super-oxidized solutions

of hypochlorites (NaOCl) are examples of

such liquids containing also antimicrobial

activity [3]

The aim of this research was the evaluation

of antimicrobial effectiveness of bacterial

cellulose dressings chemisorbed with com-

monly used wounds irrigation agents against



Material Research was carried with use of 4

reference strains of bacteria Staphylococcus

aureus ATCC 6538 Staphylococcus aureus

ATCC 33591 Klebsiella pneumoniae

ATCC 4352 and Pseudomonas aeruginosa

ATCC 15442 and 2 clinical strains of each

examined species Bacterial cellulose was

produced by Komagataeibacter xylinus

ATCC 53524 All strains come from the

collection of the Department of Pharma-

ceutical Microbiology and Parasitology The

tested antimicrobial agents were octenidine

(Octenilinreg Shuumllke) polyhexanid (Pron-

tosanreg BBraun) and super-oxidized

solution of hypochlorites (Microdacynreg

Kikgel)

Methods To evaluate antimicrobial action

of tested compounds the minimal inhibitory

concentration (MIC) and minimal biofilm

eradication concentration (MBEC) tests

were carried out To evaluate antibacterial

action of BC dressings saturated with tested

compounds the modified disc diffusion

method was performed

Results

In the MIC test the strongest action of

PHMB and OCT on staphylococci and the

weakest against P aeruginosa was obser-

ved NaOCl did not show any bactericidal

activity in the tested concentration range

Under the experimental conditions PHMB

had the strongest effect on all strains except

P aeruginosa for which OCT was better

Tested agents acted much weaker on the

biofilm than on the planktonic forms OCT

was most effective against biofilm created

by staphylococci PHMB on biofilm created

by K pneumoniae while NaOCl did not

show the biofilm eradication ability at all

None of the tested agents had an activity

against the biofilm formed by P aeru-

ginosa

In the modified diffusion-disc method in

which BC chemisorbed with analysed

compounds was applied PHMB and OCT

were effective against all tested bacterial

strains while no growth inhibition zone

around the BC disc chemisorbed with

NaOCl solution was observed PHMB

turned out to be the most active compound

in the experimental conditions

Conclusions

Bacterial cellulose is a suitable material for

dressings of antibacterial activity Antimic-

robial agents are released from the BC

without the loss of effectivity The active

substances dilution in BC does not signi-

ficantly affect activity of PHMB and OCT

PHMB and OCT have widely proven

antimicrobial activity which is confirmed

by the presented research

According to data provided here NaOCl did

not show any bactericidal activity even at

the highest concentration (undiluted) Our

results are contrary to other scientific

reports concerning matter discussed These

102

discrepancies may be result of differences in

testing models used by our vs other teams

More research is needed to draw final

conclusions on the antibacterial activity of

NaOCl [45]

Research was performed by means of

statutory funding SUBD 23020002 and

funding for young researchers

STMD23020053

References [1] Portela R Leal C et al Microbial

Biotechnology vol 4(7) 568-610 2019 DOI

1011111751-791513392

[2] Sulaeva I Henniges U et al Biotechnology

Advances vol 33(8) 1547-1571 2015 DOI

101016jbiotechadv201507009

[3] Bartoszewicz M Banasiewicz T et al Forum

Zakażeń vol 10(1) 1-30 2019 DOI 1015374

FZ2019002

[4] Herruzo R Herruzo I Journal of Hospital

Infection 2020 DOI 101016jjhin202001013

[5] Vachhrajani V Khakhkhar P Science of Wound

Healing and Dressing Materials p 43ndash47 2020

DOI 101007978-981-32-9236-9_4


Hutchinson-Gilford Progeria Syndrome therapy with RNA

interference approach

Volha Dzianisava1 Katarzyna Piekarowicz

1 Magdalena Machowska

1 Ryszard

Rzepecki1


Background

Hutchinson-Gilford progeria syndrome

(HGPS) is an extremely rare genetic dis-

order caused by a point mutation in the

LMNA gene (C1824T) This gene codes for

lamin A and C proteins however the

mutation localizes in lamin A coding

sequence Both lamin A and C are structural

proteins of the nuclear envelope which

provide mechanical support to the nucleus

De novo point mutation in exon 11 of lamin

A rises new splice site in mRNA Thereby

mature mRNA lacks part of the coding

sequence Such mRNA codes for the shorter

version of lamin A called progerin

After the synthesis in the cytoplasm lamin

A C-terminus is farnesylated This post-

translational modification enables transport

into the nucleus where anchors protein

to the inner nuclear membrane At the next

step of the maturation farnesylated

C-terminus is cleaved by endoprotease

it releases a mature form of lamin A

The progerin lacks signaling sequence

recognized by endoprotease For this reason

it accumulates into the nucleus remaining

attached to the nuclear membrane This in

turn results in the progeroid cell phenotype

ndash nuclear envelope disruption loss of

peripheral heterochromatin and abnormal

gene signaling

Nowadays effective treatment for HGPS

doesnt exist The therapy is limited to

moderation of the symptoms such as

atherosclerosis prevention of stroke and

myocardial infarction Preclinical therapies

are focused mainly on progerin levels

reduction using autophagy induction farne-

sylation inhibition or aberrant splicing

downregulation

In our study we were aimed to develop the

therapy which would decrease the synthesis

and accumulation of progerin in the nuclear

envelope We used the RNA interference

approach to specifically downregulate

progerin expression The set of siRNAs

103

sequences was designed to recognize the

junction between exon 11 and 12 in pro-

gerin mRNA but not lamin A One sequence

was tested in combination with a clinically

approved drug for HGPS therapy ndash lona-

farnib


HeLa cells were transduced with retro-

viruses to overexpress GFP GFP-lamin A

or GFP-progerin Transduced sublines were

next transfected with designed siRNAs The

efficiency of siRNA to downregulate

GFP-progerin level was indicated by the

measurement of fluorescence intensity with

flow cytometry Results were confirmed

with western blotting analysis and fluores-

cence microscopy

One of the selected siRNAs sequences was

tested in combination with lonafarnib and

analysed with the same methods

Results

Designed HGPS cellular model based on

HeLa cells was an effective tool to fast and

easy siRNAs sequences screening Two

aspects were taken into the account during

the selection ndash siRNA efficiency and

specificity

Designed siRNAs were able to reduce the

level of GFP-progerin up to 25 as flow

cytometry results showed Obtained results

were confirmed by western blotting analysis

and fluorescent microscopy

We investigated the effect of a combination

of siRNA and lonafarnib treatment as expec-

ted no antagonistic effect was observed


The accumulation of progerin in the nuclear

envelope cause defects in nuclear envelope

structure and functions thus the reduction

of progerin level seems to be crucial

for effective progeria treatment

Our results shows the sufficient decrease of

progerin level in transduced HeLa cell line

without affecting the lamin A level Besides

an additive effect of the combination of

siRNA and lonafarnib treatment lets

us consider combined therapy for further

study with patients fibroblasts cells

Among different preclinical HGPS treat-

ment strategies Huang et al study was

based on RNA interference approach with

shRNAs sequences One of shRNAs sequen-

ces was shown to specifically decreases the

progerin level in immortalized patients

fibroblasts However in our cellular model

this sequence showed weak efficiency

The research is supported by grant ERA-

NET-E-RARE-3IIITREATHGPS102018

from the Polish Agency NCBR

References [1] Huang S Chen L et al Human Genetics

vol 118444-50 2005 DOI 101007s00439-005-

0051-7


RNA interference approach for Hutchinson-Gilford Progeria

Syndrome therapy



1 Ryszard

Rzepecki1


Background





lamin A and C proteins however the muta-

tion localizes in lamin A coding sequence

104

Both lamin A and C are structural proteins

of the nuclear envelope they can poly-

merize providing mechanical support to the

nucleus


A coding sequence rise new splice site in

mRNA thereby mature mRNA lack part of

the coding sequence and code for the shorter

version of lamin A protein termed

progerin


A C-terminus is farnesylated Such post-

translational modification allows transport

into the nucleus and anchors protein to the

inner nuclear membrane Afterward farne-

sylated C-terminus is cleaved by endo-

protease releasing mature form of lamin A

and enabling its polymerization

The progerin lacks signaling sequence reco-

gnized by endoprotease For this reason it

accumulates into the nucleus remaining





gene signaling

Currently there is no effective treatment for

HGPS The therapy is limited to moderation

of the symptoms such as atherosclerosis

prevention of stroke and myocardial infar-

ction Preclinical therapies are focused

mainly on reducing progerin levels using

autophagy induction farnesylation inhibi-

tion or aberrant splicing downregulation

In our study we aimed to develop the the-

rapy which would decrease the synthesis


envelope We used RNA interference appro-

ach to specifically downregulate progerin

expression The set of siRNAs sequences

was designed to recognize the junction of

exon 11 and 12 of progerin mRNA but not

lamin A One of the most effective sequen-

ces was tested in combination with a clini-

cally approved drug for HGPS therapy

ndash lonafarnib






efficiency of siRNA to downregulate GFP-

progerin level was indicated by the mea-

surement of fluorescence intensity with flow

cytometry Results were confirmed with

western blotting analysis and fluorescence

microscopy




Results






specificity







of siRNA and lonafarnib treatment as

expected no antagonistic effect was

observed





of progerin level seems to be crucial for

effective progeria treatment





siRNA and lonafarnib treatment lets us

105

consider combined therapy for further study

with patients fibroblasts cells




shRNAs sequences One of shRNAs

sequences was shown to specifically decre-

ases the progerin level in immortalized

patients fibroblasts However in our cellular

model this sequence showed weak

efficiency




References [1] Huang S Chen L et al Human Genetics vol 118444-50 2005 DOI 101007s00439-005-051-7


Little fish big case zebrafish as a model of human metabolic disease

Magdalena Elias1 Joanna Niedbalska-Tarnowska

2 Marta Migocka-Patrzałek

1

Małgorzata Daczewska1

1Department of Animal Developmental Biology Institute of Experimental Biology Faculty

of Biological Sciences University of Wroclaw Sienkiewicza 21 50-335 Wroclaw Poland 2Laboratory of Molecular and Cellular Immunology Department of Tumor Immunology

Hirszfeld Institute of Immunology and Experimental Therapy Polish Academy of Sciences

Poland

Background

The zebrafish (Danio rerio) small aquarium

fish is a model organism used in research

concerned on human diseases since zebra-

fish genome shows high similarity with

humans [1] One of such example is the case

of human metabolic McArdle disease

caused by muscle form of glycogen

phosphorylase (PYGM) deficiency PYGM

is an enzyme which attend in the spread of

glycogen in the first step of glycogenolysis

Mutation in the PYGM gene leads to auto-

somal recessive McArdle disease in humans

Patients suffer from muscle aches cramps

and fatigue during physical exercise Such

symptoms occur due to lack of available

glucose in the muscles So far no efficient

treatment has been found

The aim of our research was to determine

if zebrafish could be an animal model for

human McArdles disease


To get a better look at the role of muscle

glycogen phosphorylase in zebrafish we

knockdown the pygm using a morpholino

technique We also use behavioural tests

and statistical analysis

Results

Our previous observations indicated that

pygm gene knockdown performed with

morpholino oligonucleotides leads to

morphological changes mimicking the

symptoms of McArdle disease [2]

Here we show the effect of Pygm knock-

down on zebrafish physical performance

The results of behavioural assay shows that

indeed the Pygm deficiency in zebrafish

decreases its motility


Our results confirmed our previous assump-

tion that zebrafish could be a good model

organism to investigate the human McArdles

disease

Acknowledgement

This work was supported by National

Science Centre (grant no 201701XNZ4

00093) and the Polish State Committee for

Scientific Research Project No 1068S

IBE2018

106

References

[1] Plantieacute E Migocka-Patrzałek M Daczewska M

Jagla K (2015) Model organisms in the fight against

muscular dystrophy lessons from drosophila and

Zebrafish Molecules 20(4)6237-53 doi

103390molecules20046237

[2] Migocka-Patrzałek M Lewicka A Elias M

Daczewska M (2020) The effect of muscle glycogen

phosphorylase (Pygm) knockdown on zebrafish

morphology The Int J Biochem Cell Biol 118

doiorg101016jbiocel2019105658


Effect of electroporation on the immunogenicity of murine Lewis lung

carcinoma cells

Magdalena Geneja1 Natalia Anger-Goacutera

1 Jagoda Mierzejewska

1 Katarzyna

Węgierek1 Agnieszka Szczygieł

1 Julita Kulbacka

2 Joanna Rossowska

1

1 Hirszfeld Institute of Immunology and Experimental Therapy Polish Academy of Sciences

Wrocław Poland 2 Department of Molecular and Cellular Biology Faculty of Pharmacy

Wroclaw Medical University Wrocław Poland

Background

Tumor cells have developed various

mechanisms that allow them to escape from

immune surveillance One of them is that

tumor cells are poorly recognized by im-

mune cells

The aim of the study was to evaluate if

electroporation of cancer cells entails chan-

ges in tumor immunogenicity


Murine Lewis lung carcinoma (LLC) cells

were electroporated using nano- or micro-

second pulsed electric field The effec-

tiveness of electroporation defined by cell

membrane permeabilization efficacy was

evaluated using propidium iodide uptake

assay performed shortly after the electro-

poration

To determine changes in immunogenicity of

LLC cells after electroporation the MHC

class I expression on the surface of electro-

porated LLC cells was examined using flow

cytometry

Moreover their influence on the differen-

tiation of dendritic cells and indirectly on

the activation of specific antitumor response

was further determined

Results

Selected electroporation parameters caused

effective permeabilization of LLC cell

membranes without induction of cell death

It was observed that some of selected electro-

poration parameters especially microsecond

electric pulses induced increased expres-

sion of MHC class I on the surface of LLC

cells Moreover dendritic cells cultured in

the presence of electroporated cells were

characterized by higher expression of MHC

class II and costimulatory antigens than

dendritic cells cultured in the presence of

control LLC cells Mature dendritic cells

were more effective in the activation of anti-

LLC immune response


Certain electroporation conditions induced

an increase in immunogenicity of LLC cells

thereby improving their recognition by

dendritic cells Based on the obtained results

we conclude that electroporation could have

an application in the preparation of DC-

based anticancer vaccines


Centre Poland (project no 201830E

NZ500711)


107

Polymers as the catalysts for nucleation and growth stable

and metastable cocrystal polymorphs

Anna Gołkowska1 Bożena Karolewicz

1 Karol P Nartowski

1

1Department of Drug Forms Technology Faculty of Pharmacy Wroclaw Medical

University Borowska 211 Wrocław Poland

Background

Pharmaceutical cocrystals are the subject of

interest in academic and industrial research

as they offer better control over physic-

chemical mechanical and pharmacokinetic

properties of active pharmaceutical ingre-

dient (API) while its therapeutic activity

remains intact This class of materials as

well as single component pharmaceutical

solids are prone to exhibit the different

packing arrangements and molecular confor-

mations within the crystal lattice with the

same chemical composition (polymorphism)

In this work we use polymer assisted grinding

(POLAG) as a mechanochemical method to

control nucleation and growth of stable and

metastable cocrystal polymorphs1


Two cocrystals known to exist in at least

two polymorphic forms were selected (1)

theophylline (TP) with benzamide (BZ) and

(2) nicotinamide (NCT) with malonic acid

(MA) The milling experiments were

performed using ball-grinder (FRITSCH

Mini-Mill PULVERISETTE 23) Excipients

used as cocrystallisation catalysts PEG (of

varying molecular weight) SPAN 80

TWEEN 20 TWEEN 80 12-penthanediol

propylene glycol Brijreg 93 Pluronicreg L-35

and Pluronicreg L-31

Structure of obtained cocrystals were invest-

tigated using X-ray powder diffraction

(PXRD) and Fourier Transform Infrared

Spectroscopy (FTIR) Thermal transitions

of cocrystals and physical mixtures of APIs

coformers and excipients were assessed

using differential scanning calorimetry

(DSC)

Results

Both polymorphic forms of TPBZ (11)

cocrystal were obtained by neat and liquid

assisted grinding (NG and LAG) as reported

previously2 Mechanochemical synthesis of

a TPBZ cocrystal using all tested excipients

resulted in formation of polymorph I

In the other investigated system NCTMA

(21) all grinding procedures produced

exclusively the NCTMA form I with no

trace of form II3

The DSC analysis of selected excipient

mixtures with APIs and coformers enabled

us to better understand the effect of polymer

addition on the cocrystallisation process


Polymorphic screening of a given compound

(cocrystal) is an important and integral step

of new drug form development Complete

knowledge of solid-state properties enables

to make a decision on most suitable poly-

morph that should be used in further

development to prevent unwanted structural

changes during the formulation and storage

of the final product

POLAG method could be applied as

a method of screening for polymorphic

forms of cocrystals Moreover used in low

quantities polymers could possibly act as

cocrystallization rate accelerating agents

The use of pharmaceutical excipients with

desired technological properties may enable

to control the properties of a final product

eg drug release tabletting properties

hydrophilicity

108

References [1] Hasa D Schneider G et al Angewandte

Chemie - International Edition 54(25) 7371-7375

doi101002anie201501638

[2] Belenguer A M Lampronti G I et al

Journal of the American Chemical Society 140(49)

17051-17059 doi101021jacs8b08549

[3] Lemmerer A Adsmond D A et al Crystal

Growth and Design 13(9) 3935-3952 doi101021

cg4006357

Fig1 Schematic representation of (A) a cocrystal (B) and (C) API polymorphs

Fig2 Molecular motfis of the (A)TPBZ form I (B)TPBZ form II (C)NCTMA form I

(D)NCTMA form II


Escitalopram affects hypocretinsorexins transmission

in hypothalamus of stressed rats

Miłosz Gołyszny1 Monika Paul-Samojedny

2 Michał Zieliński

1 Tomasz Ludyga

1

Ewa Obuchowicz1

1Department of the Pharmacology Faculty of Medical Sciences in Katowice Medical

University of Silesia 2Department of the Medical Genetics Faculty of Pharmaceutical

Sciences in Sosnowiec Medical University of Silesia

Background

Growing evidence from pre- and clinical

studies suggests that hypocretinsorexins

transmission plays a role in the patome-

chanism of psychiatric disorders (eg

anxiety and depression) Furthermore this

transmission is involved in mechanism of

action of SSRIs (eg escitalopram) The

main mechanism of action of escitalopram

consists of 5-HTT inhibition and regulation

of HPA axis activity It is known that this

109

drug alters neurotrophic factors action in the

limbic system Further may affect some

neuromodulators (eg galanin oxytocin

vasopressin and corticotropin releasing

factor)


Maternal separation (MS) was used as a

model of depression and anxiety Pups of

Wistar rats were maternally separated from

2-15P for 6h per day (between 9am and

3pm) In the adulthood (25 months) males

from stressed or control groups were assign-

ned to saline or escitalopram exposure

Drugs (10mgkg ip) or saline were admini-

strated once daily for 21 days The rats were

sacrificed 24h after the last dose of drug

Dissected brains were homogenized in

TriZol Orexins system was evaluated by

RT-qPCR method

Statistical analysis was evaluated by ΔCt

method and t-student test All the estima-

tions performed using GraphPad 704

Results

OX-A mRNA relative expression

In control + escitalopram group relative

expression factor indicated [R]=4 in

stressed rats [R]=5 and in stressed +

escitalopram [R]=4

OX1R mRNA relative expression

In statistical significant comparison was

observed [R]=4 in control + escitalopram

group in stressed rats [R]=35


Early-life stress induced up-regulation of

orexin-A expression level in the hypo-

thalamus Chronic treatment with escita-

lopram did not alter orexin-A expression

level in the stressed rats Orexin receptor 1

expression level slightly enhanced in the

stressed rats Interestingly both orexin-A

and orexin receptor 1 expression level

increased in the non-stressed rats The

activity of hypocretinsorexins transmission

might be modulated by stress and

escitalopram

References [1] Roque S Mesquita A R Palha J A Sousa

N amp Correia-Neves M (2014) The behavioral

and immunological impact of maternal separation

a matter of timing Frontiers in behavioral

neuroscience 8 192

[2] OMahony S M Marchesi J R Scully P

Codling C Ceolho A M Quigley E M amp

Dinan T G (2009) Early life stress alters

behavior immunity and microbiota in rats

implications for irritable bowel syndrome and

psychiatric illnesses Biological psychiatry 65(3)

263-267

plt001 plt00005 plt00001 two-tailed t-student

110

plt005 plt0001 two-tailed t-student


Downregulation by IL-33 of MapErk signalling pathway in gastric

epithelial cells in response to H pylori as a potential mechanism

of controlling inflammatory response

Weronika Gonciarz Agnieszka Krupa Magdalena Chmiela

University of Lodz Faculty of Biology and Environmental Protection Department

of Immunology and Infectious Biology Labolatory of Gastroimmunology

Background

Infection with H pylori Gram-negative

bacteria causes gastritis or gastric ulcer in

humans results in the gastric barrier

damage Interleukin (IL)-33 is a proinflam-

matory cytokine that alerts the host immune

system in response to a homeostasis

disorder

Aim

In this study we asked whether IL-33 is

upregulated in gastric barrier cells exposed

to H pylori components and whether controls

the MapErk (mitogen-activated protein

kinasesextracellular signal-regulated kinases)

signaling pathway


Primary gastric epithelial cells and fibro-

blasts of Caviae porcellus sensitive to H

pylori infection nontransfected or trans-

fected with IL-33 siRNA were exposed in

the cells cultures in vitro to H pylori anti-

genic complex glycine acid extract (GE)

The level of IL-33 beforeafter siRNA IL-33

tranfection of cell was measured the cell

culture supernatants by the ELISA test

(MyBiosource) Furthermore cells were

stained with anti-IL33 antibody FITC

conjugated (ThermoScientific) and imaged

in the confocal microscope (Leica SPE)

Similarly activation of Erk was evaluated

by staining the cells with FITC conjugated

antibodies to phosphorylated Erk (Cell

Signaling) The fluorescence intensity was

measured using the Victor 2 reader (Wallac)

at the wavelengths 495thinspnm (excitation) and

519thinspnm (emission)

Results


blasts non-transected with IL-33 siRNA

when treated with H pylori GE produced

significantly increased amount of IL-33

111

as compared to control cells By comparison

cells tranfected IL-33 siRNA control or GE-

induced were not able to produce IL-33

The level of phosphorylated Erk in IL-33

siRNA-silenced cells treated with GE was

significantly higher than in nontransfected

cells These results indicate that IL-33

controls the activation of MapErk signaling

pathway


Down regulation by IL-33 of H pylori-

induced Erk activation in gastric tissue cells

may be an important mechanism protecting

the gastric barrier of the host from loss of

homeostasis and the development of

excessive inflammatory response related to

the infection


Downregulation of MUC5AC production by BCG-onko mycobacteria in in vivo model of Helicobacter pylori infection

Weronika Gonciarz1 Maciej Chyb

12 Agata Tomaszewska

12 Magdalena Chmiela

1

1University of Lodz Faculty of Biology and Environmental Protection Department of Immunology and Infectious Biology Labolatory of Gastroimmunology 2Students Scientific Association of Microbiology and Immunology Institute of Microbiology Biotechnology and Immunology Faculty of Biology and Environmental Protection University of Lodz

Background Helicobacter pylori is an etiological agent of chronic gastritis duodenal ulcers and gastric cancer Colonization of gastric epithelial cells is mediated by H pylori adhesins with host mucins including mucin 5 (MUC5AC) The M bovis mycobacteria present in BCG-onko vaccine are known to posses the immunomodulatory activity This vaccine is widely used for immunotherapy of bladder cancer Due to increasing anti-biotic resistance of H pylori alternative methods of elimination of these infection are being considered We asked whether BCG-onko vaccine mycobacteria given to guinea pigs sensitive to H pylori infection are able to modulate MUC5AC production in gastric tissue which potentially may diminish Hpylori colonization

Material and Methods Himalayan Cavia porcellus male or female (500-800g) were used as a model of Hpylori infection Animals were bred in the Animal House at the Faculty of Biology and Environmental Protection University of Lodz (Poland) kept in cages with free access to drinking water and fed with standard chow Experiments were approved by the

Local Ethics Committee LKE9 (Decision 58ŁB452016)

Animals were inoculated per os with Hpylori CCUG17874 reference strain (1010

CFUml) in Brucella broth 3x at 2 days intervals Before or after administration of Hpylori the animals received BCG-onko mycobacteria (1times108 CFUml) (Biomed Lublin Poland) by the oral route Control animals were inoculated only with Brucella broth or BCG-onko The guinea pigs were euthanized after 7 or 28 days from last inoculation Hpylori infection was confir-med by histological staining (hematoxylin-eosin Giemsa) and molecular (PCR for cagA ureC genes) examination of gastric tissue as well as the production of anti-Hpylori antibodies In parallel the inflam-matory response was assessed

Deposition of MUC5AC in gastric tissue was evaluated using anti-MUC5AC antibody (MyBiosource USA) and FITC conjugated secondary antibody

Results In H pylori infected animals the production of MUC5AC was significantly increased after 7 and 28 days from inoculation as compared to non infected animals BCG-

112

onko mycobacteria which were given to animals alone or in combination with Hpylori significantly diminished the MUC5AC production which was correlated with downregulation of gastric tissue colonization with Hpylori (previous study[1])

Discussion and conclusions BCG-onko mycobacteria by diminishing MUC5AC production in the gastric mucosa of Cavia porcellus were able to prevent colonization of Hpylori Further study

is necessary in order to elucidate the role of BCG-onko mycobacteria in modulation of MUC5AC production

Reference [1] Gonciarz et al Upregulation of MUC5AC production and deposition of LEWIS determinants by HELICOBACTER PYLORI facilitate gastric tissue colonization and the maintenance of infection Journal of Biomedical Science (2019) 2623 httpsdoiorg101186s12929-019-0515-z


Autoantibodies crossreacting with TNFR induced in response

to Hpylori CagA protein during experimental infection in Cavia porcellus

Weronika Gonciarz1 Agata Tomaszewska

12 Maciej Chyb

12 Marcin Włodarczyk

1

Magdalena Chmiela1


Background Helicobacter pylori are Gram-negative rodes colonizing gastric epithelial cells in humans which may induce crossreacting antibodies and adverse inflammatory reac-tion due to the antigenic mimicry between bacterial and host components Bioinfor-matics analysis was used to show the similarity between Hpylori cytotoxin-asso-ciated gene A (CagA) protein and human as well as guinea pig tumor necrosis factor receptor (TNFR) Cavia porcellus which are susceptible to Hpylori infection were used to study the induction of anti-TNFR cross-reactive antibodies in Cavia porcellus in response to inoculation with Hpylori CagA positive strain

Material and Methods Serum samples were collected from control (10) or Hpylori infected animals after 7 28 and 60 days (102010) from inoculation The induction of IgM and IgG antibodies towards Hpylori antigens was determined by laboratory ELISA with the glycine acid

extract (GE) ndash complex of surface antigens or with recombinant CagA protein (IRIS Siena Italy) from the reference strain The crossreacting anti-TNFR IgG were detected by ELISA with TNFR (Sigma) or using synthetic P1 peptide versus P2 control peptide with or without a common CagATNFR sequence respectively (Lipo-pharm Gdańsk) Pro-inflammatory potential of anti-P1 IgG-P1 complexes was evaluated in complement binding assay

Results In all Hpylori infected animals both anti-GE IgM and IgG antibodies were raised The highest levels of anti-GE IgM were detected during acute whereas anti-GE IgG during chronic phase of infection 7 or 28 and 60 days from inoculation respectively Anti-CagA IgG were induced in 11 Hpylori infected animals 7 and 28 days post infec-tion (410 and 720) but not 60 days from inoculation Serum samples of anti-CagA IgG producers reacted in the ELISA with the complete TNFR (110 7 days from ino-culation 520 28 days from inoculation)

113

All Hpylori infected animals 7 and 28 days but not 60 days from inoculation responded by increased anti-P1 IgG pro-duction as compared to non-infected animals Absorption of anti-P1 IgG positive sera with heat inactivated Hpylori resulted in elimination of Hpylori-driven anti-P1 IgG Anti-P1 IgG-P1 immune complexes were able to activate complement indicating

the pro-inflammatory potential of such complexes

Discussion and conclusions During Hpylori infection CagA protein may induce antibodies crossreacting with host TNFR This may result in the main-tenance of inflammatory process due to complement dependent cytotoxicity or modulation of TNFR-dependent cellular responses


The utilization of the in silico methods for the description of structure

functions and protein-protein interactions on the example of Kirsten

rat sarcoma viral oncogene homolog (K-Ras)

Agata Goacuterska1 Dawid Przystupski

1

1Department of Molecular and Cellular Biology Wroclaw Medical University Poland

Background The most popular databases offer abundant datasets describing protein structure and function as well as proved or predicted protein-protein interactions In most cases profound in silico analysis underpin the further design of the research With that in mind data presented here has been obtained employing selected bioinformatics software available online commonly used for the description of structure functions and protein-protein interactions working on the example of Kirsten rat sarcoma viral oncogene homolog (K-Ras)

Material and Methods Information about the protein of interest was obtained from the online open-access databases UniProt SCOP Genebank GeneCands BioGrid IntAct STRING and OMIM 3D Simulation of the quarter-nary structure has been generated with RSCB PBD software Data were supported with a literature review conducted with the use of databases such as Science Direct and PubMed

Results KRAS is usually tethered to cell membranes because of the presence of an isoprene group on its C-terminal hypervariable region (HVR) whereas the catalytic G domain is localized at the cytoplasmatic site [1] The K-RAS protein is GDPGTP-binding protein that acts as an intracellular signal trans-ducer being activated by a guanine nucle-otide-exchange factor (GEF) and inactivated by a GTPase-activating protein (GAP) [2] K-Ras is involved in numerous cellular pathways including proliferation diffe-rentiation and senescence [3]

The GTPase activity of K-RAS is signi-ficantly enhanced by recruitment RasGAP Henceforward K-RAS can bind to SOS1 (representative of GEFs class) which forces the release of bound nucleotide (GDP) [4] Subsequently K-RAS binds GTP present in the cytosol and the GEF is released from ras-GTP After allosteric activation K-RAS recruits and regulates proteins essential for the propagation of growth factors as well as other cell signalings receptors like c-Raf and PI 3-kinase K-RAS is also involved in MAPK pathways Moreover K-RAS

114

influences gene expression ia through positive regulation of NF-kappaβ trans-cription factor activity [5-10] While wild-type K-Ras protein plays a vital role in normal tissue signaling mutated genes are potent oncogenes Mutation at the active site has been identified in about 20 of human cancers such as lung adenocarcinoma mucinous adenoma ductal carcinoma of the pancreas and colorectal cancer [11]

Discussion and conclusions K-Ras has been proven to be involved in numerous crucial cellular pathways Mutated protein isoforms behave differently presu-mably due to differences in the C-terminal hyper-variable regions While highly recur-rent in cancer attempts to target these RAS mutants with inhibitors have not been successful and has not yet become common practice in the clinic With that in mind non-direct approaches targeting crucial protein-protein interactions of K-Ras are worth attention in further research highlighting the role of thorough in silico analysis

References [1] Hancock J F amp Parton R G Biochem J vol

389 1-11 2005 DOI 101042BJ20050231

[2] Li G amp Zhang X C J Mol Biol vol 340

921-932 2004 DOI 101016jjmb200406007

[3] Karnoub A E amp Weinberg R A Nat Rev

Mol Cell Biol vol 9 517 2008 DOI

101038NRM2438

[4] Bourne H R Sanders D A et al Nature vol

349 117-127 1991 DOI 101038349117a0

[5] Rolland T et al Cell vol 159 1212-1226

2014 DOI 101016jcell201410050

[6] Ardito C M et al Canc Cell vol 22 304-317

(2012) DOI 101016jccr201207024

[7] Reid T S Terry K L et al J Mol Biol vol

343 417-33 2004 DOI 101016jjmb2004

08056

[8] Bigenzahn J W et al Science vol 362 1171-

1177 2018 DOI 101126scienceaap8210

[9] Fritsch R et al Cell vol 153 1050-63 2013

DOI 101016jcell201304031

[10] Alvarez-Moya B Loacutepez-Alcalaacute et al Onco-

gene vol 29 5911-22 2010 DOI 101038

onc2010298[11] OMIM database omimorg

Entry190070 [Online]

Fig 1 Protein-protein interactions network generated with STRING software

positive negative unspecified


115

Liposomal formulation of curcumin for human pancreatic cancer therapy

Aleksandra Grzeszczak Magdalena Zaremba-Czogalla Adrianna Zygmunt Jerzy

Gubernator

Department of Lipids and Liposomes Faculty of Biotechnology University of Wrocław

Poland

Background

Pancreatic cancer is one of the leading cause

of cancer death worldwide The survival

rate is poor and depends on general patientrsquos

health Currently the only realistic and

possible treatment for fully recover is

surgical resection followed by chemo-

therapy Due to unclear early symptoms

most of patients are diagnosed with an

advanced stage or metastasis when resection

option is impossible The pancreatic micro-

environment is very heterogenic It is cha-

racterized by high amount of extracellular

matrix components such as hyaluronic acid

collagen or fibronectin Because of that the

diffusion of the drug into tumor is limited

Over the last few years attention of rese-

archers is focused on natural substances

with anticancer properties delivered by nano

systems One of biologically active compound

is curcumin which demonstrate anti-inflam-

matory aniti-oxidant anti-proliferate and

pro-apoptotic properties Curcumin can

modulate multiple signal cascades and

pathways such as p53 pathway STAT path-

way or EGFR signaling pathway Moreover

curcumin acts as inhibitor of nuclear

transcription factor (NF-κB) what can lead

to increase of chemo-sensitivity of cancer

cells The anti-cancer potential of curcumin

cannot be fully utilised due to its physico-

chemical properties such as hydrophobic

character and low bioavailability The aim

of this study was to increase solubility and

uptake of curcumin The cytotoxic effect of

obtained liposomes with encapsulated

curcumin were tested on pancreatic cell

lines (BxPC-3 AsPC-1) and normal cell

lines (NHDF)


Curcumin liposomes were prepared by

passive loading method using extrusion

technique Curcumin and lipids (DPPC

SMDSPE PEG-2000) were dissolved

in chloroform Curcumin was mixed with

20 mg of lipids in glass tube in weight ratio

110 respectively Large MLVs were

extruded through 200 nm Nucleopore

polycarbonate filters in water bath at 64oC

Then liposomes were centrifuged at 13000

RPM for 3 minutes The size and polydisp-

ersity were determined using a Zetasizer

Nano ZS Concentration of curcumin was

measured photometrically at λ = 425 nm

The lipid concentration was determined

using the Stewart assay protocol based on

the ability of phospholipids to form a com-

plex with ammonium ferrothiocyanate The

cytotoxic effect of liposomal formulations

were assessed by the MTT assay Investi-

gated pancreatic cell lines AsPC-1 BxPC-3

and normal cells NHDF were seeded into

96-well culture plates All the lines were

treated with 0-40 uM of curcumin loaded

liposomes and incubated for 72 hours The

absorbance of the samples was calorimetri-

cally measured at 560 nm with the reference

wavelength of 630 on a microplatereader

Results

The lipid composition of tested liposomes

were DPPCSM DSPE PEG-2000 in molar

ratio 633205 The obtained liposomes

were in range from 130 to around 140 nm

with polydispersity index ranging from

0039 to 0049 The efficiency of incor-

poration of curcumin was around 50

Tested liposomal formulation showed

potential anti-cancer activity on both

116

pancreatic cell lines BxPC-3 and AsPC-1

and were less toxic to a normal cell line

(NHDF) Liposomal drug delivery system

improve bioavailability and circulation time

of curcumin


Our work demonstrated cytotoxic effect on

tested pancreatic cell lines with lower toxic

respond on normal cells These results

suggest that curcumin has higher tendency

to accumulate in tumor cells than normal

cell lines It can be explained that curcumin

binds to many proteins involved in pan-

creatic cancer Moreover curcumin inhibit

production of type I and III of collagen

which occurs in high amount in investigated

type of cancer [2] Curcumin is well docu-

mented compound which shows multiple

actions on mutagenesis or apoptosis Lipo-

somes as a drug delivery system may

increase the bioavailability stability and due

to small size of particles accumulation of

curcumin in tumor cells This study suggest

that curcumin loaded liposomes could be

a promising approach of treatment for

highly aggressive pancreatic cancer

References [1] FC Rodrigues NV Anil Kumar et al

European Journal of Medicinal Chemistry vol

177 76-104 2019 doiorg101016jejmech

201904058

[2] GP Nagaraju L Benton et al International

Journal of Cancer vol 145 10-19 2019 doi

101002ijc31867


If SiO2 nanoparticles are hemocompatibility for red blood cells in vitro

Grzywacz K1 Solarska-Ściuk K

1 Adach K

2 Włoch A

1 Męczarska K

1

Fijałkowski M2 Bonarska-Kujawa D

1

1Department of Physics and Biophysics Wrocław University of Environmental and Life

Sciences Wrocław Poland 2Laboratory of Metamaterials Institute for Nanomaterials

Advanced Technologies and Innovation Technical University of Liberec Czech Republic

Background

Mesoporous silica nanoparticles (MSNs) are

propitious candidates for nanoscale drug

delivery systems due to their unique

characteristics including biodegradability

changeable pore size mesoporosity and

high drug loading capacity SiO2 nano-

particles gain considerable attention as

competent safer and effective drug delivery

vehicles due to their mechanical chemical

and thermal characteristics The goal of this

studies is to present the hemocompatibility

evaluation of silica nanoparticles using red

blood cells a procedure which is a widely

welcomed test to ensure the safety and

compatibility of MSNs with biological

systems [1 2]


The hemocompatibility and cytotoxic effects

of SiO2 were determined after exposure to

different concentrations (0-200 microgml) at 2h

and 24h The hemolytic and osmotic resis-

tance assays were described by Cyboran et

al (2012) with a minor modification [3]

The hemolytic activity of the compounds

was determined on the basis of the concen-

tration of hemoglobin that was released

from erythrocytes after treatment with silica

nanoparticles In the osmotic resistance

assay a red blood cell suspension contain-

ing SiO2 nanoparticles at 20 microgml and 50

microgml concentration was used The impact

of nanoparticles on the shape of erythro-

cytes was determined using an optical

microscope

117

Results

Mesoporous silica nanoparticles are safe for

red blood cells in appropriate concentra-

tions Up to the concentration of 50 microgml

and 2h time of incubation they show good

hemocompatibility On the other hand after

24h incubation of erythrocytes with silica

nanoparticles the increase of hemolysis

process and decrease of osmotic resistance

of red blood cells was observed The shape

of erythrocytes was changed after treatment

red blood cells with SiO2 NPs from native

biconcave disc (discocytes) to echinocytes


Hemocompatibility is mainly an in vitro test

performed to evaluate the chances of test

samples to cause unfavorable effects on red

blood cells (hemolysis) To achieve the

desired results sample nanomaterials need

to come in direct contact with cells and tissues

That is why the safe use of nanoparticles

towards cells and tissues is the main prob-

lem in the nanodrug delivery system It is

comonly known that red blood cells are the

first component in blood coming into direct

contact with nanomaterials (because of its

size and administration route) In case if the

adverse effect of silica nanoparticles takes

place among circulating erythrocytes it

would be not important whether nano-

materials capable of transfer and freeing

up pharmaceutical ingredients are used [1]

The results showed that SiO2 nanopartciles

(particle size lt 20 nm) are safe for

erythrocytes in concentration up to 50 microgml

and show good hemocompatibility which

makes them promising materials for future

Acknowledgements

This work was supported from funds of the

statutory activities of the Department of

Physics and Biophysics Wroclaw Uni-

versity of Environmental and Life Sciences

grants no B010004119

References [1] Subhankar M Hanitrarimalala V et al RSC

Advances 9 35566-35578 2019 DOI 101039

c9ra06127d

[2] Bharti C Nagaich U et al Int J of Pharm

Invest 5124-133 2015 DOI 1041032230-

973X160844

[3] Cyboran S Oszmiański J et al Cell Mol Biol

Lett 17 77-88 2012 DOI 102478s11658-011-

0038-4


Fullerenes as targeted delivery systems used in antitumor therapy

D Gula1 E Kiełbasa

1 M Kepinska

2

1Students Scientific Association at Department of Biomedical and Environmental Analyses

Faculty of Pharmacy Medical University of Wroclaw Poland 2Department of Biomedical

and Environmental Analyses Faculty of Pharmacy Medical University of Wroclaw Poland

Anticancer drugs are cytotoxic for cance-

rous cells as well as the healthy ones

Cytostatic therapy may entail toxic effects

and be inconvenient for the patient There-

fore to increase the effectiveness and

overcome the adverse effects targeted

cancer therapy is used One of the possible

methods of selective antitumor treatment is

placing the drug in a nanocarrier [1] That

procedure improves the pharmacokinetics of

the drug increases accumulation at the site

of uncontrolled cancer and also reduces the

side effects of the drug [2]

The aim of this work was to gather infor-

mation about promising role of fullerenes in

the cancer treatment

Fullerenes are an example of organic nano-

particle which can be used as a delivery

system The most abundant synthetic

allotropic carbon form is the fullerene form

118

C60 It is a spherical particle with free space

inside characterized by a large ratio of

surface to volume C60 reacts easily with

molecules containing electrons and is also

able to bind to other compounds through

chemical physical and electrical interactions

[3] According to studies conjugations of

fullerenes with the drugs such as metho-

trexate cisplatin doxorubicin and paclitaxel

exhibit potential applications in cancer

therapy Besides the enhanced efficacy of

the cytostatics those carbon nanoparticles

also demonstrate the antitumor properties

themselves Fullerenes work by producing

reactive oxygen species (ROS) that cause

cancer cell death along with destroying

ROS sensitive linkers resulting in the

release of the drug and its action at the

target site

Along with the development of science and

chemotherapy many drug carriers have

been tested but fullerenes are the most

prominent contenders due to their properties

and structure [4] It is believed that fulle-

rene drug delivery systems could be used as

innovatory approach for treatment of cancer

References [1] Din FU Aman W et al International Journal

of Nanomedicine vol 12 p 7291-7309 2017

doi 102147IJNS146315

[2] Skivka LM Prylutska SV et al Cancer

Nanotechnology vol 9 nr 8 2018

doi 101186s12645-017-0034-0

[3] Prylutska S Grynyuk I et al Archives

of Toxicology vol 93 p 1213-1226 2019

doi 101007s00204-019-02441-6

[4] Kazemzadeh H Mozafari M Drug Discovery

Today vol 24 nr 3 p 898-905 2019

doi 101016jdrudis201901013


Is it important which form of vitamin B12 to choose during

supplementation

Viktoria Hawryłkowicz1 Natalia Komorniak

2 Adam Wojcieszonek

1

1Licenciate of dietetics Pomeranian Medical University in Szczecin 2Master of dietetics

Pomeranian Medical University in Szczecin

Background

Vitamin deficiencies are a common problem

all over the world including cobalamin

especially among those particularly at risk

of low serum levels (vegans alcoholics

patients with Castle factor deficiency)

Cobalamin is a vitamin that takes different

forms depending on the ligand attached to

the cobalt ion in the molecule This ligand

may be a cyano (- CN) hydroxyl (- OH)

methyl (- CH3) group as well as 5-deoxy-

adensine Although MeCbl and AdCbl have

two different metabolic fates different

functions and both are necessary there is

a paradigm for treating vitamin B12 defi-

ciency only with methylated form In

addition the majority of scientific research

are works using only methylcobalamin The

aim of the study was to review the literature

on metabolism persistence and absorption

of various forms of vitamin B12


The study analyzed selected papers public-

shed in Pubmed databases in 1997-2019 By

typing cobalamine 12044 items were

displayed of which limited to the most

useful 18 English-language sources

Results The literature to date reports that the best treatment for cobalamin deficiency in addition to intramuscular administration is supplementation with both active forms at the same time However other sources say that regardless of what form they took orally each of them is reduced to free form in the metabolic process In addition MeCbl

119

and AdCbl are less stable against oxidation and temperature while CnCbl is resistant to high temperatures At the same time there are reports that MeCbl is more efficiently utilized in the body compared to CnCbl

Discussion and conclusions The absorption of vitamin B12 takes place equally after consumption Regardless of whether the vitamin is taken as methylco-balamin or cyanocobalamin each of the cobalamin forms is reduced to the free form which is Cbl and cannot reach its desti-nation ie mitochondrion or cytosol unchanged The exception to this rule may be cases of mutation of the gene encoding MMACHC which prevents metabolism of MeCbl CnCbl or AdCbl The thermal stability of CnCbl is higher compared to other forms which makes it more attractive in terms of fortified food products On the other hand the study by Mayer et al Showed that methylcobalamin improved the concentration and quality of sleep of non-smoking patients who were not on a vege-tarian diet faster than more effectively than cyanocobalamin

In conclusion the form of cobalamin taken does not seem to matter among healthy people (without MMACHC gene mutation) because each of the forms undergoes a reduction reaction under the influence of the MMACHC enzyme to a free form for further use Treatment of vitamin B12 defi-ciencies with solely methylated cobalamin is therefore a controversial issue and requires more thorough scientific analysis

References The most important were listed

[1] Antony AC Vegetarianism and vitamin B-12 (cobalamin) deficiency Am J Clin Nutr July 1 200378(1)3-6 [2] Moll R Davis B Iron vitamin B 12 and folate Medicine (Baltimore) April 201745(4)198-203 [3] Shipton MJ Thachil J Vitamin B12 deficiency ndash A 21st century perspective Clin Med April 201515(2)145-50 [4] Gumprecht J Długaszek M Niemczyk A Pyryt M Olszańska E Gubała M et al Should you be concerned about vitamin B12 deficiency during metformin treatment Diabetol Practice 2016 2(6)225-9 [5] Antony AC Vitamin B12 (Cobalamin) and Folate Deficiency W Lazarus HM Schmaier AH redaktorzy Concise Guide to Hematology Cham Springer International Publishing 2019 s 37-48 [6] Wilson SMC Bivins BN Russell KA Bailey LB Oral contraceptive use impact on folate vitamin B₆ and vitamin B₁₂ status Nutr Rev October 201169(10)572-83 [7] Berenson AB Rahman M Effect of hormonal contraceptives on vitamin B12 level and the association of the latter with bone mineral density Contraception November 201286(5)481-7 [8] Obeid R Fedosov SN Nexo E Cobalamin coenzyme forms are not likely to be superior to cyano- and hydroxyl-cobalamin in prevention or treatment of cobalamin deficiency Mol Nutr Food Res 201559(7)1364-72 [9] Koyama K Usami T Takeuchi O Morozumi K Kimura G Efficacy of methylcobalamin on lowering total homocysteine plasma concentrations in haemodialysis patients receiving high‐dose folic acid supplementation Nephrol Dial Transplant May 1 200217(5)916-22


The influence of Abacavir on bone metabolism ndash preliminary results

Agata Heinrich1 dr Agnieszka Matuszewska

2 dr hab Maria Rutkowska

2

1Pharmacy student Wroclaw Medical University Wroclaw Poland 2Department

of Pharmacology Wroclaw Medical University Wroclaw Poland

Background

In the recent years the length and the quality

of life of patients infected with Human

Immunodeficiency Virus (HIV) has incre-

ased due to development of antiretroviral

therapy (ART) However simultaneously

120

other health issues occurred one of which is

the higher risk of lower bone mineral

density (BMD) and osteoporosis Initiating

ART has been shown to be the risk factor of

foregoing[1] Hence the great demand for

examining the influence of drugs used in

ART on bone metabolism


Research was conducted on fourteen male

Wistar rats They were randomly subdivided

into two groups each consisting of seven

rats Once daily group A received Abacavir

60 mgkg body weight and group C (control

group) received normal saline After eight

weeks the blood was drawn to conduct

laboratory tests

Results

After 8 weeks of the study no differences in

the serum level of N-terminal propeptide of

type I procollagen (PINP) osteoclast-deri-

ved tartrate-resistant acid phosphatase form

5b (TRACP 5b) total calcium inorganic

phosphorus nor creatinine were observed

between both study groups (PINP 374 plusmn

129 ngmL vs 418 plusmn 145 ngmL TRACP

5b 138 plusmn 013 UL vs 149 plusmn 037 UL

total calcium 989 plusmn 012 mgdL vs 989 plusmn

023 mgdL inorganic phosphorus 750 plusmn

110 mgdL vs 721 plusmn 088 mgdL

creatinine 030 plusmn 011 mgdL vs 034 plusmn

008 mgdL respectively)


Administration of abacavir for 8 weeks may

not have negative impact on bone turnover

in rats

References [1] BoneKEy Reports 4 Article number 636 (2015)

| doi101038bonekey20153


Pancreatic surgery ndash a complex interdisciplinary problem

Monika Hejnowicz 1 Weronika Mazurek

1

1Medicine Wrocław Medical University

The pancreas is one of the most important

organs in the human body It secretes

hormones that regulate the work of the

whole organism It is responsible for the

digestion of proteins and fats In addition

it controls blood glucose levels In recent

years we have seen an increase in the

number of patients with pancreatic problems

The most severe are cancers

According to data presented by the United

European Gastroenterology Association

pancreatic cancer is the 12th most common

cancer worldwide and the 7th leading cause

of cancer-related deaths worldwide Addi-

tionally The American Cancer Society

reports that in 2020 in the United States

about 57600 people will be diagnosed with

pancreatic cancer and about 47050 people

will die because of it Pancreatic cancer

accounts for about 3 of all cancers in the

US and about 7 of all cancer deaths [1]

Surgical removal of the pancreas plays

a key role in the treatment process since it

is impossible to remove the tumor without

surgical intervention It is also important

that the patient undergoes surgery at an

early stage of the disease There are two

types of surgery that can be used for such

cancers potentially curative surgery or

palliative surgery

The first one is used when the clinical tests

suggest there is a possibility to resect the

cancer The second one is used in situations

when the cancer is too widespread and the

operation is to relieve symptoms or prevent

major complications

The removal can be complete or partial and

then it only affects individual parts of the

121

pancreas head body or tail The difficulty

of the procedure is related to the location of

the pancreas and the anatomical proximity

of important organs and structures such as

the portal vein liver aorta or duodenum

Pancreatectomy involves the removal of

surrounding tissues In case of pancreatic

head cancer the most commonly used is

Whipple procedure which involves remo-

ving the pancreas head along the surro-

unding duodenum the distal part of the bile

duct together with the gallbladder and the

pyloric part of the stomach [2] When the

tumor is located in the tail or body of the

pancreas the removal affects not only these

parts of the pancreas but also the spleen

Therefore the result of surgery can be dys-

function not only of the pancreas itself but

also of nearby organs and tissues Typical

consequences of complete or partial pancre-

atectomy include deficiencies in endocrine

or exocrine pancreatic function requiring

replacement of insulin or digestive enzymes

Surgery patient preparation and postope-

rative care is not only a surgical challenge

but requires the cooperation of many spe-

cialists Such a procedure is burdened with

many major complication [4] As an impor-

tant endocrine and exocrine organ the

secretory pancreas is both an endocrine and

gastroenterological problem

References

[1] httpswwwcancerorgcancerpancreatic-

cancer aboutkey-statisticshtmlwritten_by


cancertreatingsurgeryhtml

[3] httpswwwzwrotnikrakaplleczenie-raka-

trzustki-aktualne-wytyczne

[4] httpswwwgcmplindexphpbaza_uslug-

usluga-33-operacja_z_powodu_guza_trzustkihtml


Assessment of interaction isopropyl isothiocyanate and doxorubicin

in human breast cancer cells

Sylwia Hołub1 Julita Kulbacka

2 Nina Rembiałkowska

2

1Faculty of Pharmacy Wroclaw Medical University 2Department of Molecular and Cellular

Biology Faculty of Pharmacy Wroclaw Medical University

Background

Moringa Moringa Oleifera has many

therapeutic properties Available studies

show its cholesterol-lowering anti-bacterial

anti-fungal and anti-inflammatory effects

It is suggested that it is a good source of

antioxidants thanks to which it is to prevent

the development of cancer diabetes and

heart disease Chemical analysis has shown

that the extract of this plant contains many

vitamins minerals amino acids and fatty

acids that have a beneficial effect on human

health For this reason it is used in the

kitchen and cosmetology


In the study we focused on its cytotoxic

properties To this end we analyzed the

cytotoxic effect of doxorubicin and iso-

propyl isothiocyanate (Sigma) which can

be isolated from Moring Oleifera The

concentration of isopropyl isothiocyanate

were from 0001 to 01 but doxorubicin

were from 02 to 20 microgml As a model

in vitro we used MCF-7WT cells (breast

cancer) We used the MTT test to assessed

cytotoxicity after 48 and 72 hours

Results

The results obtained show the cytotoxic

properties of isopropyl isothiocyanate against

human breast cancer cells

122


Even though the preliminary results are

promising further studies are need

Acknowledgments This research was

supported financially by the Subsidy Funds

of Department of Molecular and Cellular

Biology SUBD26020009


Analysis of multiple risk factors for negative andor indeterminate

results of anti-Tick-borne encephalitis virus antibodies in human

serum

Marta Janik 1 Sylwia Płaczkowska

2 Mieczysław Woźniak

3 Iwona Bil-Lula

4

1EUROIMMUN POLSKA Sp z oo Wroclaw 2Diagnostics Laboratory for Teaching and

Research Wroclaw Medical University 3Department of Medical Laboratory Diagnostics

Division of Clinical Chemistry Wroclaw Medical University 4Division of Clinical

Chemistry and Laboratory Hematology Wroclaw Medical University

Background

Tick-borne encephalitis virus (TBEV) infec-

tions has been the cause of the threatening

outbreaks for many years In most patients

infection results in full recovery However

in 20-75 of them neurological symptoms

has been reported The infection may lead to

meningitis encephalitis myelitis central

nervous system injury and fatal outcome [1]

Apart from several physical and chemical

manners to prevent tick bites active vacci-

nation of people highly exposed to infection

is still the most important strategy of pre-

vention Since basic vaccination does not

provide the full immunization several

booster doses are recommended (once every

five years in subjects under 60 years old and

once every 3 years in subjects over 60 years

old) [2] However in some subjects the

lack or low response to TEBV antigens is

observed

The aim of the current study was to assess

the prevalence of non-positive results for

anti-TEBV antibodies and the risk factors

for wining immunity


2315 vaccinated subjects from the high risk

group for TEBV infections participated in

this study All subjects were informed about

the aim of the study and gave written

consent for the participation The study

protocol was approved by the Bioethics

Committee of Wroclaw Medical University

(Poland) Data about age sex and vacci-

nation were acquired from written question-

naire due to participants` memory Clinical

samples of whole blood (to obtain serum)

were collected Serum aliquots were stored

at -20degC before analysis Commercial

ELISA test was used for anti-TEBV IgG

serum level assessment and results were

defined as negative for values of lt120

VIEUml indeterminate for values between

120 VIEUml and 165 VIEUml and

positive for values of 165 VIEUml or more

Results

Data showed that 862 of subjects who

underwent vaccination were positive for

anti-TEBV antibodies within 5 years As

much as 138 of subjects underwent to

basic or basic and remaining vaccination

were not protected barely after vaccination

The analysis of time dependent frequency of

non-positive results of anti-TEBV antibo-

dies concentration (lt165 VIEUml) showed

that time elapsed since the last dose of basic

vaccination was associated with increased

123

number of negative results for anti-TEBV

antibodies (Chi2 for trend p=0012) Data

showed that the serum titer of anti-TEBV

antibodies decreased during the time since

the vaccination In 273 of subjects vacci-

nated ge4 years before testing and as much as

143 of participants underwent vacci-

nation less than 1 year before testing anti-

TEBV titer did not reach 165 VIEUml As

much as 75 of subjects under 60

vaccinated lt1 year before testing and 90

of subjects vaccinated ge4 years before

testing had negative or indeterminate titer

(lt165 VIEUml) of anti-TEBV antibodies

A logistic regression showed that longer

time since the vaccination doses constantly

increased the odds ratio (OR 1206

95CI1100-1324) for non-positive values

of anti-TEBV antibodies when a higher

number of booster constantly decrease the

odds ratio (OR 0573 95CI 0498-0660)

for non-positive values of anti-TEBV

antibodies


Some previous studies suggest that some

subjects who received vaccination did not

response to vaccination and some congenital

disorders might be a main cause of vacci-

nation failure [3] Severe cases of TBE has

also been observed in patients previously

vaccinated with initially proper response to

vaccination [4] For this reason non-respon-

ders or low responders are not protected

after primary infection or vaccination and

lower titers of neutralizing antibodies delay

the clearance of the virus and may result in

infection of neuronal cells [5] This study

demonstrates that vaccination schedule

should be personalized The extension of the

interval of booster immunization is risky

and all subjects should be surrounded by

care consisting of more frequent monitoring

of serum antibodies by individual schedule

to adjust the frequency of subsequent doses

of booster vaccination

References [1] Zajkowska J Czupryna P Forum Zakażeń vol

443-5212013

[2] Stefanoff P Rosińska M et al Przegl

Epidemiol vol 60151-159 2006

[3] Guumlnther G Haglund M et al Clin Diagn Virol

vol 8 17-29 1997

[4] Charrel RN Attoui H et al Clin Microbiol

Infect vol 101040-1055 2020 doi101111j1469-

0691200401022x

[5] Heinz FX Stiasny K et al Emerging Infect Dis

vol 1969-76 2013DOI103201eid1901120458


Innovative treatment of ischemic strokes complications based

on steam-cells therapy and cytokine administration

Michał Jarocki1 Kacper Turek

1 Michał Gebuza

1

1Faculty of Medicine Wrocław Medical University

Brain ischemic stroke is a serious medical

condition which may lead to severe sys-

temic consequences In the vast majority of

affected patients it is followed by compli-

cations that lower the quality of their life

According to statistical data from 2015

brain strokes are the primary cause of dis-

ability among the middle-age group [1]

People who overcome brain stroke report

considerable deterioration of cognitive and

motoric brain functions The range and

severity of impairment is adequate to the

areas and regions affected by ischemic

stroke Nowadays the most popular treat-

ment method is rehabilitation The aim

of the method is to restore the full capacity

of the movements and brain functions in

some cases even comparable to condition

before damage

124

However in most cases the effectiveness of

rehabilitation is not high enough to satisfy

patients and their demands The full reco-

very is rarely achieved by this traditional

treatment

The application of stem cells as well as cytokines with pro-regenerative properties gives a perspective that in the not far future patients suffering from ischemic strokersquos complications may count on more reliable methods of treatment [2] Aside from the low efficiency the stem-cells based therapy may also be less inconvenient and time-consuming This means shorter recovery times and lower costs for the patients [3] Proposed methods could be applied in the upcoming decade as a standalone or compli-mentary treatment method for the traditional physiotherapy

In this project the authors gather all the cur-rently available data about stem-cells based treatment of ischemic strokersquos compli-cations Presented data would show the perspectives of alternative to rehabilitation methods of recovery for patients suffering from stroke

References

[1] D Smajlović Strokes in young adults Epidemiology and prevention Vasc Health Risk Manag vol 11 pp 157-164 Feb 2015 [2] C V Borlongan Concise Review Stem Cell Therapy for Stroke Patients Are We There Yet Stem Cells Transl Med vol 8 no 9 p sctm19-0076 May 2019 [3] M T Turnbull A C Zubair J F Meschia and W D Freeman Mesenchymal stem cells for hemor-rhagic stroke status of preclinical and clinical research npj Regen Med vol 4 no 1 Dec 2019


Does ambient air pollutant PM10 effects on fetal growth

Marta Jędrzejak Emilia Nowiczuk

Students Scientific Society at the Department of Toxicology Faculty Pharmacy Wroclaw Medical University Wroclaw Poland

Background Theme of publication lsquoEffects of prenatal exposure to ambient air pollutant PM10 on ultrasound-measured fetal growthrdquo pointed out the problem of dependency between higher air pollution and fetal growth Most of the studies were conducted in areas when air pollution level is lower (Europe USA) and results were inconsistent The hypo-thesis that was investigated exposure that

the high level of PM10 (1408 microgm3 in the

current study) during pregnancy increases the risk of abnormal fetal growth

Material and Methods The study group consisted of 8877 pregnant women inhabited in Lanzhou China between 2010-2012 Before studies women were interviewed using a standardized and structured questionnaire to collect main

information Implemented ultrasound measu-rements (18 583) of four fetal growth parameters like biparietal diameter (BPD) femur length (FL) head circumference (HC) and abdominal circumference (AC)

Measurement of PM10 in Lanzhou have been compered with level of PM10 in Wroclaw to estimate possibility of occur-rence abnormal fetal growth in Poland

Results The results of interviews show that more than half of the women were younger than 30 (635 ) non-smoker (81 ) non-drinking alcohol (998) Most of them had a normal BMI (68 9) and didnrsquot have maternal diabetes (988) Pregnant women were used mostly gas or electricity as a cooking fuel which reduce possibility of different source of PM10

125

When the level of PM10 was exceeding over

150 microgm3 there were increases in standar-

dized FL ( P= 00012 ) and HC (P=00078) compared with lower levels Correlation

between increase PM10 (every 10 microgm3)

and standardized BPD (P=00016) were noticed HC measurement proved the highest dependency between PM10 level and abnormal fetal growth

In Wroclaw average value PM10 in 2019

was 26 microgm3


Study suggested that high level of PM10

during pregnancy increased the risk of

occurrence abnormal fetal growth (over and

under growth) Depends of the method

which was chosen there are difference

between degree of deviation from the norm

However a small amount of similar rese-

arches does not allowed to unequivocal

determining the correctness of the results

Menace of abnormal growth in Poland is not

as high as in China because the level of

PM10 is significantly lower

Preoccupation of this subject is very impor-

tant because of continuous growth of

contents PM10 in inhaled air

References [1] Effects of prenatal exposure to ambient air

pollutant PM10 on ultrasound ndash measured fetal

growth Authors Nan Zhao Jie Qiu Shuangge Ma

Yaqun Zhang Xiaojuan Lin Zhongfen Tang

Honghong Zhang Huang Huang Ning Ma Yan

Huang Michelle L Bell Qing Liu and Yawei

Zhang httpsairwroclawpiosgovpl


Do-it-yourself artificial pancreas systems (DIY APS) non-commercial

software for diabetes control improvement

Magda Kabaj1 Kamil Klimas

1 Agnieszka Święcicka-Klama

12 Zuzanna Sycz

3

1Department and Clinic of Angiology Systemic Hypertension and Diabetology Wroclaw

Medical University Wroclaw Poland 2Department of Social Medicine Wroclaw Medical

University Wroclaw Poland 3Department of Biology and Medical Parasitology Wroclaw

Medical University Wroclaw Poland

Type 1 diabetes (T1D) represents about

10 of all types of diabetes The onset of

illness is usually between the age of 10-14

T1D mainly affects children and young

people (lt30 years of age) the long-term

duration of the disease is conducive to the

occurrence of complications The goal

of diabetes management is to avoid the

development of acute and chronic diabetes

complications by maintaining glucose levels

within the recommended values [1] It is cur-

rently an incurable disease and has a huge

impact on the daily life of patients Appro-

priate motivation makes it easier to treat and

to accept the disease T1D is a disease that

requires great self-discipline from patients

but does not stop them from living a normal

life

A slowdown of diabetes complications

development can be achieved only through

better glycemic control However recent

studies indicate that a small number of

people with T1D achieve their therapeutic

goals [2] The development of medical

technology brings better and better devices

for continuous glucose monitoring (CGM)

and continuous subcutaneous insulin infusion

(CSII) systems The idea of automation the

measurement of glucose and insulin supply

and thus creating an artificial pancreas

is developing [3] While the hybrid closed-

loop system (Medtronic MiniMed 670G)

126

has been launched first to market the

community of people with diabetes and

their families united online under hashtag

bdquoWeAreNotWaitingrdquo was developing non-

commercial do-it-yourself artificial pancreas

system (DIY APS) [4] The system initially

created by few people grew in strength

Nowadays several open-source software are

available to make a closed loop by yourself

The number of users is constantly incre-

asing however the technology is unregu-

lated [34] Currently only observational

evidence exists but their results are

promising In surveys bdquoloopersrdquo reported

eg more time in target glucose range better

sleep less frequent hypoglycemia impro-

ved HbA1c or even more confidence and

fewer mood swings [5] More reliable data

is required to determine safety and outcomes

The described technology is an example of

a positive patient initiative that can improve

the quality of care but omits the tests and

regulatory steps required by the medical

industry There are many ethical and legal

doubts about the participation of healthcare

professionals in DIY APS usage At present

the software shouldnrsquot be prescribed promo-

ted initiated or recommended but due to

the spread of the system and promising data

on its effectiveness knowledge about it

should be expanded

References [1] ADA et al Diabetes care Suppl 1S43-8 2006

DOI 102337dc14-S081

[2] Foster N Beck R et al Diabetes technology amp

therapeutics 212 66-72 2019 DOI 101089

dia20180384

[3] Jennings P Hussain S Journal of Diabetes

Science and Technology 2019 DOI 101177

1932296819894296

[4] Crabtree T McLay A et al Practical

Diabetes 36(2) 63-68 2019 DOI101002

pdi2216

[5] Hng T Burren D Internal medicine journal

4811 1400-1404 2018 DOI 101111imj14105


Fluoride content in spirulina (Arthtospira spp) supplements

from conventional and organic cultivation

Kałduńska Justyna1 Kikut Justyna

1 Palma Joanna

1 Komorniak Natalia

1 Żwierełło

Wojciech2 Skoacuterka-Majewicz Marta

2 Styburski Daniel

2 Konecka Nina

3

1Department of Human Nutrition and Metabolomics Pomeranian Medical University

in Szczecin Poland 2Department of Medical Chemistry Pomeranian Medical University

in Szczecin Poland 3Department of Neurocognitive Science Pomeranian Medical

University in Szczecin Poland

Background

Spirulina (Arthrospira plantensis) is

a microalga with hypolipidemic hypogly-

caemic as well as anticarcinogenic and anti-

inflammatory properties It is a rich source

of proteins vitamins as well as micro- and

macro-nutrients which makes it an

increasingly popular dietary supplement

However there is a risk that spirulina supple-

ments may act as a source of fluoride in

human nutrition Significant intake of fluorine

and the exposure to its low concentrations

have negative effects on the human orga-

nism The aim of the study was to determine

the content of fluoride (F) in spirulina

supplements originating from conventional

and organic farming


The material used in the study was 34

spirulina dietary supplements in tablet and

powder form originated from traditional and

organic cultivation A total of 34 spirulina

samples from different countries of origin

were obtained from specialist shops F

127

concentrations in individual samples were

measured by the potentiometric method

with a fluoride ionselective electrode (Orion

9409 BN Thermo Scientific USA) The

statistical analysis was performed using Stat

Soft Statistica 130 and Microsoft Excel

2010

Results

The F content in the supplements included

in the study ranged from 10526 plusmn212 to

165805 plusmn522 ppm Fluoride content in

supplements in tablet form was significantly

higher (p= 00241) No statistically signi-

ficant differences in the F content were

observed depending on the method of

cultivation (conventional vs organic)


The study demonstrated that spirulina culti-

vation method did not have a significant

effect on fluoride concentrations and supple-

ments originating from organic cultivation

did not have statistically significantly lower

fluoride levels On the other hand a signi-

ficant difference was observed in the fluo-

ride content of supplements sold in tablet vs

powder form

References [1] A Finamore M Palmery et al Oxidative

Medicine and Cellular Longevity vol 2017

3247528 2017 DOI 10115520173247528

[2] D Ozsvath Reviews in Environmental Science

and BioTechnology vol 8 59-79 2009 DOI

101007s11157-008-9136-9


Dietary habits of children and adults

Patrycja Kapczuk1 Natalia Komorniak

2 Wojciech Żwierełło

3 Marta Skoacuterka

3

Daniel Styburski3

1Department of Biochemistry Pomeranian Medical University in Szczecin Powstańcoacutew

Wlkp 72 St 70-111 Szczecin Poland 2Department of Human Nutrition and Metabolomics

Pomeranian Medical University in Szczecin Broniewskiego 24 Str 71-460 Szczecin

Poland 3Department of Medical Chemistry Pomeranian Medical University in Szczecin

Powstańcoacutew Wlkp 71 Str 70-111 Szczecin Poland

The recent studies show that dietary habits

of both children and adults depart from the

recommendations In comparison with other

European countries of similar socio-eco-

nomic status the number of deaths and

incidence of chronic diseases due to poor

diet in Poland is increasing The studies

published by the Developmental Period

Medicine on food products popular among

children and adolescents (between 1 and 18

years of age) list fries (27) chocolate

(27) and pizza (23) to be the most

popular The study also revealed a highly

worrying negative correlation between the

age of the children under study and the

consumption of food products containing

sugar glucose and high-fructose corn syrup

ndash all of which increase the risk of obesity

and diabetes type 2 Given the age of the

children under study the main potentially

harmful food additives are salicylates

monosodium glutamate sodium benzoate

potassium sorbate iron sulphate ammo-

nium sulphate BHT and calcium disodium

EDTA Another study conducted on adoles-

cents between 13 and 15 years of age shows

the popularity of highly processed food

products among this age group Fast-food

snacks and sweetened carbonated beverages

are consumed several times a week (33)

and several times a month (60) In turn

the consumption of sweetened carbonated

beverages (1 litre per day) was declared by

70 of the adolescents under study

128

The study on dietary habits of adults found

that the frequency of consumption of milk

brown rice whole grain noodles and rolled

oats decreases with age Conversely the

consumption of salt coffee and offal

showed an increase with age of the respon-

dents These findings were also confirmed

by anthropometric measurements as the

body mass index and the percentage of body

fat showed an increase with age According

to the data by WHO 650 million of adults

and 340 million of adolescents suffers from

obesity worldwide The statistics are wor-

rying as even among the patients with

morbid obesity awaiting bariatric surgery

the dietary habits showed numerous short-

comings Among such patients the fre-

quency of consumption of animal fats (lard

fatback) energy drinks and beer was higher

with an increase of waist-hip ratio (WHR)

Keywords additives highly processed

food nutrition

References [1] Wolski T Karwat ID Najda A (2005)

Kontaminacja i suplementacja żywności a zdrowie

Post Fitoter 1-2 35-41

[2] Kozioł-Kozakowska A Pioacuterecka B Schlegel-

Zawadzka M (2016) Wpływ postaw rodzicielskich

na sposoacuteb żywienia dzieci w wieku przedszkolnym w

Krakowie na tle uwarunkowań socjodemo-

graficznych Zdrowie Publiczne i Zarządzanie 201

12(1) 82-89

[3] Budrewicz S Banaszczak M Piotrowski J

Czerwińska M Stachowska E (2017) Allergens and

food additives including potentially harmful ones

present in food products that are preferred by

children and adolescents Dev Period Med 21

131-138

[4] Patterson ME Yee JK Wahjudi P Mao CS

Lee WP (2018) Acute metabolic response to high

fructose corn syrup ingestion in adolescents with

overweightobesity and diabetes JNIM 141-7

[5] Wanat G Grochowska-Niedworok E Kardas

M Całyniuk B (2011) Nieprawidłowe nawyki

żywieniowe i związane z nimi zagrożenie dla

zdrowia wśroacuted młodzieży gimnazjalnej Hygeia

Public Health 46 381-384

[6] Adamska E Ostrowska L Adamska E

Maliszewska K Citko A Waszczeniuk M

Przystupa W Majewski R Wasilewska A

Milewski R Krętowski A Goacuterka M (2012) Roacuteżnice

w nawykach i preferencjach żywieniowych osoacuteb

dorosłych w zależności od wieku Roczniki

Państwowego Zakładu Higieny 63 73-81

[7] Obesity and overweight [online] World Health

Organization [date of access 30072019]

Available httpswwwwhointnews-roomfact-

sheetsdetailobesity-and-overweight


KIDMED test ndash a tool for childrens diet assessment

Anna Kawalec1 Giorgia Piscitelli

2 Laura Dallolio

2 Krystyna Pawlas

1

1Department of Hygiene Faulty of Medicine Wroclaw Medical University Wrocław

Poland 2Department of Biomedical and Neuromotor Sciences University of Bologna

Bologna Italy

Background

The mediterranean diet is indicated as an

ideal dietary pattern characterized by large

intake of vegetables fruits bread and other

forms of cereal rice beans and nuts It also

includes virgin olive oil as the principal

source of fat moderate amounts of dairy

products and fish and red meat in low

amounts The mediterranean diet provides

most of the recommended macro- and

micronutrients in the right proportion as

well as antioxidants and has positive effect

on health by reducing the risk of wide range

of chronic diseases such as myocardial

infarction diabetes cancer arthritis and

other pathologies related to oxidative stress

Dietary habits are shaped in childhood and

unfavourable diet may result in negative

long-term health consequences therefore it

129

is of utmost importance to promote healthy

lifestyle among children The aim of this

study was the assessment of adherence

to mediterranean diet among primary school

children


The observational study was conducted

among school-age children from a primary

school in Wałbrzych The study group con-

sisted of 75 children (45 boys and 30 girls)

mean age was 8 years old plusmn 11 months

Under supervision of teachers and parents

children completed a 7-day dietary diaries

by colouring and describing composition of

their diet for a whole week and for every

meal

Obtained data were analysed with the use of

the KIDMED test with a presumption that

mediterranean diet can be treated as a model

of a healthy and well-balanced diet regard-

less geographical region

The KIDMED test (Mediterranean Diet

Quality Index for children and teenagers) is

a tool to evaluate the adherence to the medi-

terranean diet for children and youths and

consists of 16 yes or no questions (Table 1)

For each yes response one point is given

to answers representing positive food habits

and one point is subtracted for those

representing negative food habits Three

categories of adherence were defined

poor ndash score le 3 points

medium ndash score 4-7 points

high ndash score ge 8 points

Results

Mean scoring was 309 points the median

was 3 points In 45 children (60) the adhe-

rence to model diet was assessed as poor in

29 (3867) as medium and only in one

child as high There was no significant

difference between boys and girls

Table 1 KIDMED test questions

and scoring

KIDMED test Scoring

a fruitfruit juice every day + 1

a second fruit every day + 1

vegetables regularly once a day + 1

vegetables more than once a day + 1

fish at least 2-3 times per week + 1

fast-food more than once a week - 1

legumes more than once a week + 1

pastarice 5 or more times per week + 1

cereals or grains for breakfast + 1

nuts at least 2-3 times per week + 1

regular use of olive oil at home + 1

skipping breakfast - 1

a dairy product for breakfast + 1

commercially baked goods or

pastries for breakfast - 1

two yogurts andor some cheese daily + 1

sweets and candy several times every

day - 1


Our results indicate that diet of 60

participants was assigned as poor which

corresponds with several studies reporting

both low adherence to mediterranean diet

among children (from 21-27 to 739)

and insufficient knowledge about healthy

diet and role of fruits and vegetables con-

sumption This unfavourable trend is com-

mon to numerous countries Although the

KIDMED test is designed for mediterranean

region its usage in epidemiological studies

becomes more popular also in other Euro-

pean regions It can be also implemented in

nutrition education programs aiming to esta-

blish healthy eating habits at young age that

will have beneficial effects in later life and

in prevention strategies for reducing child-

hood overweight and obesity

130

References [1] G Cabrera H Fernaacutendez et al Nutr Hosp 2015

vol 32(6)2390-9 doi 103305nh20153269828

[2] T Grassi F Bagordo et al Int J Food Sci

Nutr2019 Oct 211-10 doi 10108009637486

20191679725

[3] A Buja G Grotta et al Eur J Pediatr 2020 Jan

17 doi 101007s00431-020-03577-9

[4] P Iaccarino Idelson L Scalfi et at Nutr Metab

Cardiovasc Dis 27(4)283-299 2017doi 101016

jnumecd201701002

[5] L Serra-Majem R Garcia-Closas Public

Health Nutr 2001 4(6A)1433-8

[6] RM Akhtar Khan E Ahmet Med Forum

Monthly 2008198-10

[7] S Sahingoz N Sanlıer et al Appetite

201157272-7


The advanced wound care device comprised of a porous matrix

of bovine tendon collagen and glycosaminoglycan

Agata Kawalec1 Krystyna Pawlas

1

1Department of Hygiene Wroclaw Medical University

Background

Burns are known to be the most devastating

injuries found in medicine Despite the huge

development in the process of wound care

the results of treatment are still not satis-

factory


The paper presents treatment options using

a matrix to regenerate the dermis

Results

The biocompatible matrix for dermis rege-

neration allows the patients body to rebuild

its own skin It comes in two forms as

a single or double layer membrane system

The first layer is composed of bovine

collagen surrounded by glucosaminoglycan

(GAG) the second layer iscomposed of

silicone The matrix after implantation in

the wound bed is colonized by host cells In

the first stage monocytes and neutrophils

produce and secrete cytokines stimulating

chemotaxis of endothelial cells forming

a network of new blood vessels Then migra-

ting fibroblasts producing and secreting

proteins and proteoglycans initiate so-called

Remodeling ndash matrix reconstruction and

reconstruction of the primitive dermis

structure At the same time the collagen

matrix is gradually biodegradable The

structure of the first matrix layer is

a scaffold for the migration of fibroblasts

macrophages lymphocytes and endothelial

cells of capillaries forming a network of

new vessels In the healing process the

collagen and GAG layers are resorbed and

the new collagen is synthesized by fibro-

blasts which lasts about 3 weeks After this

period if a well vascularized new dermis is

formed the protective silicone layer

is removed and in its place an autogenic

thin epidermal transplant is performed

whose cells expand to form mature epi-

dermis covering the entire wound surface

As a result the defect is replaced with

functional dermis and aesthetic epidermis


The use of a matrix to regenerate the dermis

is especially important in extensive burns

Despite the fact that the first surgery with

the use of matrix in Poland was carried out

some time ago because of the costs the

treatment is not available in all centers

dealing with burnt patients

References [1] M Reynolds DA Kelly et al Walker NJ

Crantford C Defranzo AJ Use of Integra in the

Management of Complex Hand Wounds From

Cancer Resection and Nonburn Trauma Hand (N

131

Y) 201813(1)74-79 doi 101177155894471

7692090

[2] DK Chang MR Louis et al The Basics of

Integra Dermal Regeneration Template and its

Expanding Clinical Applications Semin Plast Surg

201933(3)185-189 doi 101055s-0039-1693401

[3] LEM de Haas KLM Gardien et al The Use

of Integra in Extensive Full-Thickness Scalp Burn

Involving the Skull in a Child J Craniofac Surg

201930(3)888-890 doi 101097SCS000000

0000005375

[4] Y Liu AC Panayi et al Current Available

Cellular and Tissue-Based Products for Treatment

of Skin Defects Adv Skin Wound Care 2019 32

(1)19-25 doi 10109701ASW0000547412

54135b7


Fabrication of Drug loaded Micro-needles for effective muscular pain

relief

Nabeel Asif Ali Khan1 Kinjal Patel

2 Tabassum Khan

2 Munira Momin

2 Soham

Shah3 Vaibhavi Sonetha

3 Sharmi Majumdar

3

1Faculty of Chemical and Materials Engineering University of Alberta Edmonton Canada 2SVKMrsquos Dr Bhanubhen Nanavati College of Pharmacy Mumbai India 3Dwarkadas

Jivanlal Sanghvi College of Engineering Mumbai India

Background

Microneedles (MNs) have wide applications

for drug delivery as they are minimally

invasive to the skin creating small size

pathways that deliver drugs directly below

the stratum corneum without having to

penetrate the upper layers [1] This can

potentially be used to treat conditions re-

quiring instant relief or sustained effect The

minimally invasive nature of MNs improves

acceptability and patient compliance This

project proposes to use MNs comprising

a suitable combination of muscle relaxant to

give instant pain relief and NSAID to

provide sustained release [3]


We needed to fabricate microneedles with

precision up to the micrometre For this we

used two methods of making high precision

molds The first method involved using

a micro-CNC machine to make positive

molds of stainless steel The second method

involved using the Nanoscribe 3D printing

machine University of Alberta [2] The

drugs selected were diclofenac sodium and

thiocolchicoside The hyaluronic acid (HA)

dissolving MN patches were fabricated by

a two-step micromolding process The MN

master molds were used to prepare PDMS

female molds as per the manufacturers HA

aqueous solution was poured over the

female molds followed by vacuum treat-

ment The samples were dried in a sealed

desiccator overnight at room temperature

After being peeled off from the female

molds the final MN patches were sealed

and stored in the desiccators at room

temperature and protected from light The

two drugs were then incorporated into the

HA dissolving MN patches at various drug

concentrations They were subsequently

detached from the molds and stored

appropriately

Results

The first method using Nanoscribe gave us

only the top needle layer as the machine had

size limitations It included preparing data

for exposure tool loading and unloading

specimen development and post develop-

ment UV curing We also used an adhesive

to attach it to the rest of the positive mould

made on a regular CNC machine In the

second method we used a simple Micro-

CNC however the accuracy of the mould

was not upto the mark We used PDMS to

132

make the negative molds and poured the

dual drug loaded polymer solution in this

mould to develop the MNs The MNs were

evaluated for mechanical properties and

drug release profile using Franz diffusion

cell The mechanical strength showed an

inverse relationship with drug loading The

release studies indicated an initial release of

around 50-60 attributed to the water-

soluble nature of HA About 75 and 85

of drug in the MNs was released in the

medium at 30 min and 60 min respectively


We tried two ways of making the positive

molds of the MNs the accuracy was

important as every patch made should have

the same amount of drug Using the

Nanoscribe machine included an extra step

of using an adhesive giving an accurate

needle mold Using the micro-CNC ma-

chine gave us a singular block but with

lower accuracy The mechanical strength of

MNs is affected by tip radius material

composition and geometry as observed in

this study The dual drug loaded MN deli-

very system could serve as a promising

technology alternative for effective delivery

of dual drugs in the treatment of muscle

pain circumventing the gastrointestinal side

effects of NSAIDs

References [1] Waghule T Singhvi G Dubey S K Pandey

M M Gupta G Singh M amp Dua K (2019)

Microneedles A smart approach and increasing

potential for transdermal drug delivery system

Biomedicine amp Pharmacotherapy 109 1249-1258

doi 101016jbiopha201810078

[2] Kathuria H Li H Pan J Lim S H

Kochhar J S Wu C amp Kang L (2016) Large

Size Microneedle Patch to Deliver Lidocaine

through Skin Pharmaceutical Research 33(11)

2653-2667 doi 101007s11095-016-1991-4

[3] Kochhar J S Tan J J Y Kwang Y C amp

Kang L (2019) Microneedle Patch for Fast Onset

and Long-Lasting Delivery of Painkillers

Microneedles for Transdermal Drug Delivery 67-

80 doi 101007978-3-030-15444-8_5


Calcium electroperation for prostate cancer tratment ndash in vitro studies

Aleksander Kiełbik1 Olga Michel

2 Wojciech Szlasa

1 Julita Kulbacka

2 Jolanta Saczko

2



Background

Calcium electroporation (CaEP) proved its

effectiveness in numerous in vivo and in

vitro studies Currently the first clinical

trials are onging The aim of this research

is to explore the possibility of calcium EP

on prostate cancer


The Du-145 prostate cancer cells ware

elctroporated in HEPES buffer with 8 100

micros pulses of 400 to 2000 Vcm and

incubated for short time in different calcium

concentrations 0mM 05mM 1mM 2mM

5mM and 10 mM Secondly with optimized

parameters the cell electroporation with

calcium was performed The cell viability

was measured with MTT assay Cell

permeability assay with Yo-Pro-1 dye was

performed with flow cytometry The cell

motility after CaEP was investigated with

scratch assay

Results

The optimal reversible electroporation was

achieved when 800 Vcm pulses ware

applied The CaEP has significantly decre-

ased the prostate cancer cell viability

133


The research shows that CaEP can be po-

tentially used as an alternative for minimal-

invasive focal therapy of prostate cancer

Moreover we suggest that calcium ions can

potentially strengthen the effect of already

incorporated into clinical practice irrever-

sible electroporation

Fig 1 Cell viability and permeability after electroporation without drug

Fig 2 Cell viability after electoporation with calcium in different concentrations


Safety of using calcium supplements in people with casein allergy

Justyna Kikut1 Natalia Komorniak

1 Justyna Kałduńska

1 Joanna Palma

1 Paula

Halecka1 Monika Szewczyk

1 Małgorzata Sarna

1 Robert Budawski

1 Wojciech

Żwierełło2 Marta Skoacuterka-Majewicz

2 Daniel Styburski

2 Nina Konecka

3





Background

Calcium is an essential component of the

human body It is mainly absorbed in the

small intestine and is deposited in the bones

by circulation [1] One of the most impor-

tant roles of calcium in the body is skeletal

134

mineralization Adequate intake of this

element is important at every stage of human

life Normal peak bone mass achieved in

adulthood minimizes the risk of osteo-

malacia and osteoporosis later in life [2]

Groups at risk of calcium deficiency include

children and adolescents the elderly but

also people who are allergic to cows milk

proteins [3] The main milk proteins include

casein Casein increases calcium absorption

but at the same time has a strong allergenic

effect which in the group of people with

cows milk allergy may even lead to anaphy-

lactic shock [4] Patients must completely

eliminate dairy which is the best source of

calcium in the diet [3] The elimination diet

makes it necessary to take supplementation

[5] The source of calcium in dietary prepa-

rations is often not specified on the packaging

which makes it reasonable to suspect that

the calcium in these products comes from

dairy products In addition casein is resis-

tant to high temperatures and it is difficult to

remove it from all dairy products and it

may even be found in milk replacers with

a high degree of hydrolysis

The aim of the study was to evaluate that

calcium supplements available in the phar-

macy are safe for people allergic to cowrsquos

milk proteins


The study was carried out on a randomly

selected sample of 21 calcium supplements

by the enzyme-linked immunoassay method

using a casein test (kit test produced by

Neogen Noack Polen District) Tested

supplements were available without a pres-

cription Each sample was tested in two

repetitions

Results

All tested samples showed a concentration

below the methods limit of quantification

lt25 ppm These values also do not exceed

the permissible amount of casein that can

cause allergic reactions


The report of the Supreme Audit Office

showed that supplements may be conta-

minated and adulterated Manufacturers

often do not declare all substances used

to prepare the supplement Hence patients

may have various types of ailments eg

gastrointestinal which will be difficult to

identify The possibility of adulteration

or contamination of the supplement indi-

cates the need to conduct the above tests

regularly in order to maintain the highest

safety of such preparations in patients [6]

The calcium supplements tested appear to

be safe for use by people with a casein

allergy Each time the amount of casein in

the samples was below the detection

threshold

References [1] Li K Wang X et al Clin Interv Aging vol

132443-2452 2018 DOI 102147CIAS157523

[2] Vannucci L Fossi C et al Nutrients vol

10(12)1930 2018 DOI 103390nu10121930

[3] Hodges J Cao S et al Nutrients vol

11(4)718 2019 DOI 103390nu11040718

[4] Hochwallner H Schulmeister U et al

Methods vol 66(1)22-33 2014 DOI 101016

jymeth201308005

[5] Kansu A Yuumlce A et al Turk J Pediatr vol

58(1)1-11 2016 DOI 1024953turkjped

201601001

[6] Raport of Supreme Audit Office ndash Admission to

trading supplements 2017


Nanomedicine for targeted treatment of tumor diseases

Rene Kizek1 Karel Sehnal

12 Marta Kepinska

3 Carlos Fernandez

4 Dominik

Banaacuteš2 Dagmar Uhliacuteřovaacute

2 Martina Staňkovaacute

2 Michaela Všetičkovaacute

2 Branislav

Ruttkay-Nedeckyacute1 Božena Hosnedlovaacute


3

135

1Department of Human Pharmacology and Toxicology VFU Brno Palackeacuteho třiacuteda 19461

612 42 Brno Czech Republic 2Department of Research and Development Prevention

Medicals sro Tovaacuterniacute 342 742 13 Studeacutenka-Butovice Czech Republic 3Department of

Biomedical and Environmental Analyses Wroclaw Medical University Wroclaw Poland 4School of Pharmacy and Life Sciences Robert Gordon University Aberdeen United

Kingdom

Background

Cancer is the second leading cause of death

in developed countries It is known that

standard antitumor therapy has a number of

serious adverse biological effects One of

these is a lack of selectivity for tumor tissue

resulting in significant side effects The

relatively low therapeutic concentration of

the active compound often results in drug

resistance and multi-resistance of tumor

cells


This review analysis (2000-2020) is focused

on nanotechnology The methodology of the

choice used scientific studies from more

than 500 viewed articles from databases

MEDLINE PubMed and Google Scholar

based on the search phrases such as

nanomedicine thiols nanoparticles

Results and Discussion

Nanotransporters for targeted treatment are

a modern and effective way of personalized

approach [1] Carbon gold silver and other

nanoparticles (NPs) can be used as the basis

of the nanotransporter [2] NPs can enter a

cell independently of its type and functional

group attached to the surface of the nano-

particle Various in vitro and in vivo studies

have shown that many functionalized

nanoparticles are biocompatible [3] The

physico-chemical properties of nanoparti-

cles play a decisive role in their potential

toxicity [4] For NPs shorter and thicker

nanotubes have been found to exhibit lower

toxicity Chemically functionalized NPs are

much better water-soluble and have greater

stability in the physiological environment

Attempts to use NPs to target multivalent

ligands in cancer are increasing rapidly [5]

In addition to passive targeting methods

based on the enhanced permeability and

retention (EPR) effect and the specific acidic

environment in the tumor strategies for

actively targeting a selected tumor using

ligands or antibodies that increase the speci-

ficity of the nanotransporter are also investi-

gated However a protein corona plays

a major role in the application of NPs

in vivo [6 7] A protein corona is a cluster

of all proteins that can bind to NPs Protein

corona formation is usually associated with

a significant reduction in therapeutic poten-

tial Albumin is the most abundant compo-

nent of the protein corona It has been

shown that the composition of the protein

corona depends on the structure and physico-

chemical properties of the NPs However

the effect of surfactants on the structure of

NPs on the composition and formation of

the protein corona has not yet been

investigated In our experiments the effect

of the interaction of serum albumin and NPs

was studied A completely unanswered

question is the interaction of nanoparticles

with thiol compounds such as low-mole-

cular-weight glutathione or metallothionein

In addition to the above Giulimondi et al

observed increased expression of albumin

receptors in some malignant tumors (liver

gallbladder but also breast cancer)7 Protein

corona-modified nanoparticles may be useful

for targeting albumin receptor-overex-

pressed tumor cells

Conclusions

This research area of nanomedicine is

completely open and will certainly bring

many unexpected discoveries in the near

future

136

Acknowledgements

The work was carried out with the support

of the H2020 CA COST Action (CA15114)

and International Collaboration Project of

The European Technology Platform for

Nanomedicine

References [1] H S Leong K S Butler et al Nat

Nanotechnol vol 14 629-635 doi101038

s41565-019-0496-9 (2019)

[2] J Nam S Son et al Nat Rev Mater vol 4

398-414 doi101038s41578-019-0108-1 (2019)

[3] F Peng M I Setyawati et al Nat Nanotechnol

vol 14 279-280 doi101038s41565-018-0356-z

(2019)

[4] X D Xue Y Huang et al Nat Commun vol

9 Article number 3653 (3615) doi101038

s41467-018-06093-5 (2018)

[5] S A Chechetka Y Yu et al Nat Commun vol


ncomms15432 (2017)

[6] X Lu P P Xu et al Nat Commun vol 10

Article number 4520 (4514) doi101038s41467-

019-12470-5 (2019)

Fig 1 The negative impact of the protein corona on the nanoparticle targeting ability

A target protein (blue) a native protein (red green)


Cryoglobulins and their damaging effect on organs Etiopathogenesis symptoms and diagnosis of cryoglobulinemia

K Klimas1 M Kabaj

1 A Święcicka-Klama

12 Z Sycz

3

1Department and Clinic of Angiology Systemic Hypertension and Diabetology Wroclaw Medical University Wroclaw Poland 2Department of Social Medicine Wroclaw Medical University Wroclaw Poland 3Department of Biology and Medical Parasitology Wroclaw Medical University Wroclaw Poland

Cryoglobulinemia is a rare disease (1 case per 100000 individuals) consisting in antibodies which precipitate in vitro at low temperatures and disappear when incubated at 37˚C [1] It is observed in the course of various disorders Three types of cryo-globulinemia are identified on the basis of laboratory investigations type I with monoclonal immunoglobulins (IgG IgM IgA or their κ or λ light chains strongly connected with monoclonal gammopathies such as a monoclonal gammopathy of undetermined significance (MGUS) or a B-

cell lineage malignancy) type II (most often) with a mixture of a monoclonal IgM with rheumatoid factor (RF) activity and polyclonal IgG (linked to hepatitis C virus infection) and type III with a mixture of polyclonal IgM with rheumatoid factor activity and polyclonal IgG (connected with autoimmune diseases like SLE) [2 3]

Cryoglobulins cause tissue damage in the mechanism of increasing blood viscosity (hyperviscosity syndrome) plugging and thrombosis of blood vessels (deposits

137

in small arteries and capillaries) and deposition on the epithelium of blood vessels and the blood complement acti-vation (the systemic vascular inflammatory reaction) [4] Each type may be manifested by a different set of symptoms Type I cryoglobulinemia is characterized mainly by the presence of skin lesions such as purple-colored papulae on lower limbs livedo reticularis Raynaudrsquos phenomenon ulcers and tissue necrosis Types II and III more frequently present with symptoms within other systems with strong muscle and joint pains becoming more intensive at lower temperatures peripheral polyneuropathy hepatic dysfunction respiratory symptoms or membranoproliferative glomerulitis [15]

Diagnosis is based on clinical and labo-ratory findings The detection of serum cryoglobulins is necessary for correct classification It involves a blood test in which the sample must be kept at 37degC for a period of time before being cooled After precipitation at 4degC and centrifugation measurement of cryocrit can be performed Further steps (washing and prewarming of precipitate electrophoresis immunofixation at 37degC) help to type

cryoglobulins [2 3] Treatment depends on underlying disease and includes eg immu-nosuppressors corticosteroids andor plasmapheresis

The aim of this paper is to present etio-pathogenesis clinical features diagnostic approach and treatment of cryoglobuli-nemia Presented information may help clinicists diagnose this very rare disease

References [1] Takada S Shimizu T et al Cryoglobulinemia (Review) Molecular Medicine Reports 201263-8 httpsdoiorg103892mmr2012861 [2] Muchtar E Magen H Gertz MA How I treat cryoglobulinemia Blood 2017129289-98 httpsdoiorg101182blood-2016-09-719773 [3] Overview of cryoglobulins and cryoglo-bulinemia ndash UpToDate nd httpswwwuptodate com contentsoverview-of-cryoglobulins-and-cryoglobulinemia (accessed February 14 2020) [4] Ostojic P Jeremic IR Managing refractory cryoglobulinemic vasculitis challenges and solutions Journal of Inflammation Research 20171049-54 httpsdoiorg102147JIRS114067 [5] Ferri C Mixed cryoglobulinemia Orphanet Journal of Rare Diseases 2008325 httpsdoiorg1011861750-1172-3-25


Synthesis of new pyrrolo[34-c]pyrrole derivatives with potential analgesic properties

T Kłosiński1 K Chmiel

1 A Redzicka

2

1Student Scientific Club of Chemistry of Drugs Wrocław Medical University Borowska 211 50-556 Wrocław Poland 2Department of Chemistry of Drugs Wrocław Medical University Borowska 211 50-556 Wrocław Poland

Background Cyclooxygenases (COX) is a group of enzymes that play a large role in a bio-synthesis of prostaglandins (PGE) and thromboxane (TXA2) COX catalyses the conversion of arachidonic acid into those substances Arachidonic acid derivatives have diverse physiological functions such as causing inflammation blood clotting pain or fever [1]

COX have two different isoforms COX-1 is responsible for maintaining the proper function of internal organs so inhibiting it may cause adverse effect such as kidney damage gastrointestinal bleedings and ulcers COX-2 stimulates PGE biosynthesis in inflammatory cells Therefore it is crucial to develop Non-steroid Anti-Inflammatory Drugs (NSAIDs) selective towards COX-2 izoenzyme [2]

138

N-Mannich bases are promising group of chemical compounds with potential anal-gesic properties and high pharmacological activity [3] In this paper we are presenting synthesis of new pyrrolo[34-c]pyrrole deri-vatives which may present those properties

Material and Methods The key substrates for the synthesis of the final compounds derived from the pyr-rolo[34-c]pyrols were imides (butyl or phenyl substituent) These imides were obtained by process of multistage synthesis the first stage of which was condensation that leads to obtaining diester Next conden-sation of diester with anilinen-butylamine was performed Intermediate product was then transformed into diacid during hydro-lysis Anhydride diaccid was converted into amide-acids Finally amide-acids under-went intramolecular cyclization with the formation of imide The imide constituted key substrate for the subsequent stage of synthesis [3]

In a reaction of the proper imide with formal-dehyde arylopiperidine derivatives (in ethanol) final compounds (Fig1) were obtained

Results Initially In silico computations were con-ducted to calculate estimated bioactivity Molecular docking procedure was perfor-med based on Lamarckian Genetic Algorithm (LGA) using AutoDock 42 program [4]

Derivatives with butyl or phenyl group (R1) attached to nitrogen atom in pyrrole group and with 4-chloro or 4-bromo (R2) phenyl group attached to piperidine were obtained General formula of these new compounds is presented on Fig 1

The compounds were obtained with a very good efficiency (70-80) Structures of those compounds were confirmed by FTIR and 1H NMR

Discussion and conclusions Derivatives obtained during this synthesis may be promising path in further research on selective COX-2 inhibitors but further tests are required to determine their phar-macological properties Therefore due to prospective results of Autodock compu-tations and good efficiency of synthesis we decided to send these compounds to In vitro cyclooxygenase (COX-1 and COX-2) inhibition assay and evaluation of viability

References [1] R Dubois S Abramson et al FASEB Journal vol 12 (12) p 1063-1073 September 1998 DOI 101096fasebj12121063 [2] A Blobaum L Marnett et al Journal of Medicinal Chemistry vol 50 (7) p 1425-1441 7 March 2007 DOI 101021jm0613166 [3] A Redzicka Ł Szczukowski et al Bioorganic amp Medicinal Chemistry vol 27 (17) p 3918-3928 1 September 2019 DOI 101016jbmc2019 07033 [4] Morris G M Hueyet al J Computational Chemistry 2009 16 2785-91 DOI 101002 jcc21256

Figure 1 General formula of obtained compounds

R1 = -C4H9 -C6H5 R2 = -Cl -Br


139

Expression of selected inflammatory parameters as markers

of endometriosis progression ndash pilot study

Izabela Kokot1 Agnieszka Piwowar

2 Marcin Jędryka

34 Ewa Maria Kratz

1


of Pharmacy Wroclaw Medical University Poland 2Department of Toxicology Faculty

of Pharmacy Wroclaw Medical University Poland 3Department of Oncology Clinic

of Gynaecological Oncology Faculty of Medicine Wroclaw Medical University Poland 4Department of Gynaecological Oncology Lower Silesian Cancer Centre Poland

Background

Endometriosis is a gynaecological disease

that pathogenesis seems to be strict asso-

ciated with inflammatory processes To

evaluate the subclinical inflammation in the

blood serum of women with endometriosis

we were tested C-reactive protein (CRP)

immunoglobulin G (IgG) interleukin 1β

(IL-1β) interleukin 6 (IL-6) and Human

Chitinase-3-like Protein 1 (YKL-40) to find

a non-invasive marker of endometriosis

progression


The study group of patients consisted of 43

women with histologically confirmed endo-

metriosis The serum samples ndash material for

the study - were collected at the Department

of Gynaecological Oncology in Lower

Silesian Cancer Centre (Poland) Based on

the revised American Fertility Society

(rAFS) classification 20 women displayed

moderate and 23 severe endometriosis

(stage III and IV respectively) We also

included 19 women with no history of

endometriosis as a healthy control group In

all samples we measured concentrations of

CRP IgG IL-1β IL-6 and YKL-40

Statistical analysis was performed using

Statistica PL version 133 (StatSoft Inc

Tulsa OK USA) The p value lt 005 was

considered significant

Results

We observed a significant higher concen-

tration of CRP in patients with IV stage of

endometriosis in comparison to III stage of

endometriosis (p=0021) No significant

differences were found in the concentration

of serum IgG IL-1β IL-6 and YKL-40

between III and IV stage of endometriosis

however we observed lower median

concentrations of IgG and YKL-40 and

higher median concentration of IL-1β and

IL-6 in patients with severe endometriosis

as against moderate stage We observed

significant difference in concentrations of

CRP IgG IL-1β and IL-6 between group of

severe endometriosis and the control group

(p=0005 p=0016 p=0014 plt0001

respectively) whereas we did not observe

any differences between III stage of

endometriosis and control group There was

a positive correlation between CRP and IL-1β

(r=034 plt0001) CRP and IL-6 (r=058

plt0001) and a negative correlation between

IgG and IL-6 (r=-042 plt0001)


The symptoms of endometriosis are often

non-specific and may suggest many various

gynaecological diseases One of the reason

of diagnostic delay of endometriosis confir-

mation is trivialization the symptoms of

disease not only by patients but also by

gynecologists Unfortunately the laboratory

medicine has not yet a noninvasive biomar-

kers that may diagnose andor confirm the

stage of endometriosis progression It is

clearly seen that diagnosis of endometriosis

requires special algorithm with adequate

medical procedure with specific biomarkers

140

One of the panels of such inflammatory

biomarkers could be serum CRP IgG IL-6

and IL-1β which can support the diagnostics

of endometriosis what is especially impor-

tant in the stage of the disease

References [1] S As-Sanie R Black et al Assessing research

gaps and unmet needs in endometriosis American

Journal of Obstetrics and Gynecology 221(2)86-

94 2019 DOI 101016jajog201902033

[2] FO Dorien I Flores et al Noninvasive

diagnosis of endometriosis Review of current

peripheral blood and endometrial biomarkers Best

Pract Res Clin Obstet Gynaecol vol 50 pp 72-

83 2018 DOI 101016jbpobgyn201804001

[3] CM Kyama S Debrock et al Potential

involvement of the immune system in the

development of endometriosis Reprod Biol

Endocrinol vol 1 no 1 p 123 2003 DOI

1011861477-7827-1-123

[4] WC Andrews VC Buttram S J Behrman

Revised American fertility society classification

of endometriosis 1985 Fertil Steril vol 44

no SUPPL 2 pp 7-8 Mar 1985 DOI 101016

s0015-0282(16)48430-x


The importance of monitoring blood homocysteine levels in clinical

practice

Natalia Komorniak1 Justyna Kikut



2

Marta Skoacuterka-Majewicz2 Daniel Styburski

2 Joanna Palma

1 Nina Konecka

3


in Szczecin Poland2Department of Medical Chemistry Pomeranian Medical University

in Szczecin Poland3Department of Neurocognitive Science Pomeranian Medical


Background

Homocysteine (Hcy) is an amino acid that

includes thiol groups in its particles It is

formed in all cells of the organism as a result

of the metabolism of one of the exogenic

amino acids ndash methionine The necessary

cofactors for the biochemical course of

changes of Hcy include folates and vitamin

B12 (the methylation process of Hcy into

methionine) vitamin B6 (the catabolism of

Hcy into cysteine) vitamin B2 (the process

of forming 5-methyltetrahydrofolate by

means of 5 10- methylenetetrahydrofolate

reductase) betaine (the remethylation

process) magnesium and lithium (adenosine

triphosphate is present in the form of a

complex with magnesium ions and lithium

ions this compound which is high in

energy participates in the transformation

of methionine into S-adenosylmethionine

via methionine adenosyltransferase) The

aim of the study was to evaluate the impor-

tance of monitoring blood homocysteine

levels in clinical practice


The literature present in the PubMed and

Embase databases has been reviewed

Results Homocysteine undergoes autooxidation which leads to the formation of biologically active substances that participate in signa-ling pathways associated with increased cell toxicity facilitating apoptosis necrosis the formation of blood clots and the ampli-fication of oxidative stress The autooxi-dation of Hcy thiol groups results in the formation of reactive oxygen species (ROS) ie hydrogen peroxide Additio-nally by reducing the activity of glutathione peroxidase and the redox potential Hcy amplifies the effects of ROS In conse-quence lipids proteins carbohydrates and nucleic acids undergo oxidation leading to endothelium dysfunctions the damaging of

141

blood vessel walls the activation of pla-telets and the formation of blood clots Hyperhomocysteinemia is also toxic for neurons and glial cells Among other reasons the toxicity is a result of the intracellular mobilization of Ca2+ and oxidative stress within the endoplasmic reticulum leading to apoptosis the rebuilding of extracellular matrix in the brain and endothelium dysfunctions Furthermore free oxygen radicals have the ability to induce the activity of the NR1 subunit of the NMDA receptor (N-methyl-D-aspartate) which leads to its increased sensitivity to stimu-lating amino acids (glycine serine gluta-mate) resulting in disorders in the integrity of the blood-brain barrier

Discussion and conclusions In physiological conditions when vitamin B is properly supplied in the diet the concen-tration of Hcy in the blood should be correct An increase in the concentration of Hcy can often be the result of deficiencies of vitamins B6 B12 and folate Apart from an improperly balanced diet the increase of the level of Hcy in blood can be the result of diseases of such organs as kidneys or the thyroid as well as other factors such as neoplasms psoriasis diabetes the use of some drugs (eg metformin) alcohol con-sumption smoking elderly age menopause achlorhydria with a low level of Castlersquos external factor intestinal inflammatory diseases and surgeries of the digestive tract (eg bariatric surgeries) The factors that contribute to the increase of the level of

homocysteine in blood also include gene polymorphisms mainly in reference to methylenetetrahydrofolate reductase (MTHFR) ndash the key enzyme of the folate cycle An increased level of homocysteine in blood correlates with the existence of specific pathological units such as cardiovascular diseases atherosclerosis a stroke depression Alzheimerrsquos disease and osteoporosis

In conclusion it is worth to consider a routine examine of homocysteine level especially in people with diseases that co-occur with elevated homocysteine levels in the blood such as atherosclerosis and depression

References [1] Škovierovaacute H Vidomanovaacute E et al Int J Mol Sci 17(10) 1733 2016 DOI 103390ijms 17101733 [2] Boldyrev A Bryushkova E et al Curr Aging Sci 6(1) 29-36 2013 DOI 10217418746098 112059990007 [3] Petras M Tatarkova Z et al J Physiol Pharmacol 65 15-23 2014 [4] Curro M Gugliandolo A et al Neurochem Res 30 1485-95 2014 DOI 101007s11064-014-1338-7 [5] Faraci FM Lentz SR Stroke 35 345-47 2004 DOI 10116101STR00001151611064667 [6] Wu X Zhang L et al Rdox Biol 20 45-59 2019 DOI 101016jredox201809021 [7] Farina N Jernereacuten F et al Exp Gerontol 99 146-50 2017 DOI 101016jexger201710008 [8] Saito M Marumo K Curr Osteoporos Rep 2018 16(5) 554-60 2018 DOI 101007s11914-018-0469-1


Intake of protein antioxidant vitamins and cobalamin in patients after

brain tumor surgery

Nina Konecka1 Justyna Kikut

2 Natalia Przybyła

1 Marta Kęsik

1 Natalia

Komorniak2 Justyna Kałduńska

2 Joanna Palma

2 Daniel Styburski

3 Marta Skoacuterka

3

1Depertament Of Neurocognitive Science Pomeranian Medical University in Szczecin 2Department Of Human Nutrition And Metabolomics Pomeranian Medical University in Szczecin 3Department of Medical Chemistry Pomeranian Medical University in Szczecin

142

Background

Surgery is a strong stress factor that comes

with inflammation Metabolic response after

surgery is associated with use of own

energy source like proteins needed to

wound healing After operation it is

observed higher levels of glucose protein

breakdown loss of nitrogen in the urine and

negative nitrogen balance [1] The opera-

tional gesture is associated with increased

protein catabolism Depending on the

severity of the procedure the catabolic

phase may last from 5 to 10 days [2] That is

why the proper protein nutrition of patients

is so important The aim of the study was to

determine the intake of protein antioxidant

vitamins and cobalamin intake in patients

after brain surgery


The study group included 30 patients of the

Pomeranian Medical University ndash Neuro-

surgery Clinic in Szczecin admitted for

brain tumor surgery The study involved

15 women (average age 4830 plusmn 1453

years) and 15 men (average age 5633 plusmn

1467 years) Each patient underwent

a nutritional questionnaire which was intro-

duced into the 5D Diet nutrition program to

determine the level of protein and individual

vitamin intake The results obtained were

compared to the Nutrition Standards for the

Polish Population

Results

The average protein intake in both groups

was higher than recommended by the

standards The intake of vitamin B12 and

A in men exceeded the norm twice The

consumption of vitamins A C E in both

groups exceeded accepted norms


Higher protein intake in both sexes has

a positive effect on the bodys protection in

the phase of increased postoperative cata-

bolism and protein loss In addition higher

intake of vitamins with antioxidant activity

seems to have a protective effect on

inflammation occurring during and after

surgery

References [1] Nowakowski P Jurszewicz P Family

MedicineampPrimary Care Review 3313-316 2014

[2] Hasiak J Przegląd Urologiczny vol 575 2012


Light-activated antibacterial polymer coatings

Bartosz Kopyciński1 Alicja Duda

2 Marcin Staszuk


2

1Department of Engineering Materials and Biomaterials Faculty of Mechanical

Engineering Silesian University of Technology Gliwice Poland 2Department of Physical

Chemistry and Technology of Polymers Faculty of Chemistry Silesian University

of Technology Gliwice Poland

Background

Despite significant advances in medicine

nosocomial infections are still a serious

problem In Europe 45 million patients are

affected by hospital-acquired infection

every year and nearly 40 thousand of them

ends with the patients death This increases

the duration of treatment and can cause

hospitals overcrowding [1] Possible way

to protect against bacteria is using antimic-

robial surfaces One of the most promising

solution is preparation of coatings able

to produce singlet oxygen (1O2) as a ger-

micidal agent Singlet oxygen has been the

subject of research for almost 60 years [2]

It is an excited form of molecular oxygen

arising in photosensitization process which

143

is successfully used in photodynamic the-

rapy [3] Because of its ability to bacteria

killing singlet oxygen is also used for

coatings design As a result of light expo-

sition particles of photosensitizer (eg dye)

suspended in the polymer matrix generate

singlet oxygen which reduces surface level

of bacteria


Polydimethylsiloxane (PDMS (C2H6OSi)n)

provided by Dow Chemical Company

thionine (C12H9N3SmiddotC2H4O2) tert-Butyl

(TBA C4H10O 99) and methanol

(CH3OH 998) purchased from Sigma-

Aldrich were used to obtain composite

coatings PDMSThionine films were rece-

ived by solution spin-coating method

Singlet oxygen photogeneration was obser-

ved using ultravioletndashvisible spectroscopy

(UV-Vis) Measurements were made in

quartz cuvettes with laser at wavelength 532

nm and xenon lamp using respectively

13-diphenylisobenzofuran (DPBF) and

α-Terpinene (both supplied from Sigma-

Aldrich) as an oxygen indicators Scanning

electron microscope (SEM) was used to

observe the morphology of prepared

coatings

Results

During UV-Vis measurements observation

of decreasing absorbance for singlet oxygen

indicator was done at appropriate wave-

length (410 nm for DPBF and 266 nm for

α-Terpinene) which is caused by its

oxidation Both measurements lasted for 30

minutes with 5 minutes intervals In case of

DPBF oxidation process absorbance decre-

ased from 0599 to 0296 α-Terpinene

absorbance decreased from 1049 to 0933

The morphology of the imaged surfaces was

characterized by the presence of small

randomly distributed thionine agglomerates

suspended in a PDMS matrix


Obtained coatings showed good ability to

singlet oxygen generation Lower efficiency

of photogeneration for test with xenon lamp

is the result of its work in the full range

of visible light (greater range of photo-

sensitizer excitation) In the case of obser-

vation with laser photosensitizer excitation

occurs at a wavelength close to its maxi-

mum absorption therefore the photogene-

ration efficiency is higher The presence of

thionine agglomerates in the polymer matrix

may be the result of insufficiently long

mixing time of the solution (thionine didnrsquot

dissolve and disperse well) In order to

confirm the effectiveness of the developed

PDMSThionine films appropriate micro-

biological tests are planned Methods of

preventing contagions based on use of

antibacterial coatings may become in the

future an effective tool in the fight against

hospital infections

This work was supported by European

Social Fund in the framework of the project

Silesian University of Technology as

a Center of Modern Education based

on research and innovationrdquo POWR03

0500-00-Z09817

References [1] B Allegranzi S B Nejad et al The Lancet vol

377 228-241 2011 DOI 101016S0140-

6736(10)61458-4

[2] M C Derosa R J Crutchley Coordination

Chemistry Reviews vol 233-234 351-371 2002

DOI 101016S0010-8545(02)00034-6

[3] A Blacha-Grzechnik A Drewniak et al

Journal of Photochemistry and Photobiology A

Chemistry vol 388 1-22 2020 DOI 101016

jjphotochem 2019112161


144

Inflammation induced by long-term acetylsalicylic acid supple-

mentation affects density of vasoactive intestinal polypeptide-like

immunoreactive (VIP-LI) nerve fibres in the porcine jejunum

Ismena Kordylewska1 Barbara Jana

2 Jarosław Całka

1


and Mazury in Olsztyn 2Division of Reproductive Biology Institute of Animal Reproduction

and Food Research Polish Academy of Science Olsztyn

Background

Vasoactive intestinal polypeptide (VIP)

is considered to be one of the most

important substances involved in the intesti-

nal regulatory processes VIP is an inhi-

bitory factor causing the smooth muscles

relaxation suppression of the gastric acid

secretion and vasodilation of the submucosa

[1]

Acetylsalicylic acid also known as aspirin

(ASA) is a commonly used drug with anal-

gesic antipyretic anti-inflammatory and

anticoagulant effects

The aim of the present study was to deter-

mine the influence of high doses of acetyl-

salicylic acid on the enteric nerve fibres of

the porcine jejunum


This study was performed on 8 immature

female pigs of the Pietrain x Duroc breed

(approximately 8 weeks old and 20 kg body

weight) The first group consists of control

gilts (n=4) received empty gelatin capsules

orally and the second group ndash experimental

(n=4) received gelatin capsules with

acetylsalicylic acid orally 100 mgkg body

weight After 4 weeks the pigs were

euthanized Following fixation and freezing

section double immunofluorescence staining

was performed Antibodies against the

protein gene-product 95 (PGP 95) and

against the VIP were used as primary

antibodies As secondary antibodies were

used Alexa Fluor 488 and 546 Stained 14

microm sections were examined under Olympus

BX51 fluorescence microscope The evalu-

ation of VIP-LI-positive nerve fibres within

the wall of jejunum was carried out on the

basis of the counting of all VIP-LI-positive

nerve fibres per microscopic observation

field (01 mm2) Such an estimation was

carried out in five sections per animal (in

five fields per section)

Results

The present immunohistochemical studies

revealed that in the jejunum of pigs treated

with aspirin the distribution of VIP-LI nerve

fibres were significantly altered Inflam-

mation induced by long-term administration

of high doses of acetylsalicylic acid caused

increase in the density of the VIP-LI

intraganglionic nerve fibres


Our study showed that after aspirin supple-

mentation there was increased density of the

intraganglionic nerve fibres immunoreactive

to VIP-LI Similar changes were obtained

during previous studies [2 3] We can

assume that enteric nerve fibres underwent

adaptation to the induced pathological

condition Our findings of increased density

of VIP-LI-immunoreactive intraganglionic

nerve fibres as a consequence of aspirin-

induced inflammation provide valid eviden-

ce of the important function of this peptide

in neuronal responses to inflammation

References [1] McConalogue Karen and John B Furness 3

Gastrointestinal neurotransmitters Baillieres

clinical endocrinology and metabolism 81 51-76

1994 doi 101016S0950-351X(05)80226-5

145

[2] Makowska K (2018) Chemically induced inflammation and nerve damage affect the distribution of vasoactive intestinal poly-peptide‐like immunoreactive (VIP‐LI) nervous structures in the descending colon of the domestic pig Neurogastroenterology amp Motility vol 30(11) 2018doi 101111nmo13439

[3] Rytel L amp Calka J Acetylsalicylic acid-induced changes in the chemical coding of extrinsic sensory neurons supplying the prepyloric area of the porcine stomach Neuroscience letters vol 617 218-224 2016 doi 101016jneulet201602029

This study was supported by the National Science Centre (grant no 201829N NZ400348)


Thyroid carcinoma in the light of new research ndash a review

Łukasz Kotyra1 Aneta Starzec

2

1Department of Medical Biochemistry Wroclaw Medical University 2Department of Pharmacognosy and Herbal Medicines Wroclaw Medical University

Thyroid cancer is the most frequent endocrine carcinoma and the first cause of death among endocrine cancers It makes up over 95 of endocrine carcinomas[1] In the last 35 years the incidence of thyroid cancer has tripled in western European countries[2] Epidemiological research carried out in vitro as well as in vivo shows correlation between occurrence of malig-nant thyroid carcinomas and hormones Thyroid cancer is more frequent in women than men (especially in women at pro-creation age) [3] because estrogens and receptors for estrogens (ER) play an important part in genesis reprogramming and progression of the cancer

Nowadays medical science reports the existence of several types of malignant thyroid carcinomas and their classification is based on histopathological examination thus there are papillary follicular medul-lary and anaplastic (undifferentiated) thy-roid carcinomas among which papillary

follicular and anaplastic derive from folli-cular cells (90 of cases) whereas me-dullary stems from parafollicular cells (C cells) Moreover there are two more types of thyroid cancer thyroid lymphoma and thyroid sarcoma

Currently applied treatment involves surgical removal of pathological tissue radioactive iodine therapy and treatment with suppressive doses of L-T4

References [1] Tafani M De Santis E Coppola L Perrone GA Carnevale I Russo A Pucci B Carpi A Bizzarri M Russo MA Bridging hypoxia inflammation and estrogen receptors in thyroid cancer progression Biomed Pharmacother 2014 68(1)1-5 [2] Lee JC Gundara JS Glover A Serpell J Sidhu SB MicroRNA expression profiles in the management of papillary The Oncologist 2014191141-1147 [3] Boelaert K Thyroid gland revised guidelines for the management of thyroid cancer Nat Rev Endocrinol 6 (2010) pp 185-186


Optimization of the conditions of 3D bioprinting with the microextrusion

method ndash the influence of the pressure on the viability of the cells

Patrycja Kowalska1 Marta Klak

1 Magdalena Gomoacutełka

1 Paweł Turowski

1

Grzegorz Tymicki1 Piotr Cywoniuk

1 Katarzyna Kosowska

1 Tomasz Bryniarski

1

Tomasz Dobrzański1 Andrzej Berman

1 Michał Wszoła

1

1Foundation of Research and Science Development Warsaw Poland

146

Background

The 3D bioprinting with the use of live cells

is the newest technique from the field of

biomedical engineering One of the most

important points of the procedure is saving

the cells and letting them stay fully

functional in the obtained bioconstruct The

most popular method used in 3D bioprinting

is microextrusion [1] Nevertheless inde-

pendently of the used bioink dosing method

it has to be kept in mind that inside the

cartridge act forces that interact straightly

with the cells suspended into entire bioink2

While using microextrusion method with

the change of pressure we change the forces

acting on the cells In this way we may

control the conditions of cells after the bio-

printing process [4] In our work we

showed that each cell line that is used

in bioprinting process should had indivi-

dually selected pressure range [3]


Cells in the number of 5middot105mL are sus-

pended in 3 of alginate and are bioprinted

with the use of BioX bioprinter and with the

pressure in the range of 0-200kPa After

bioprinting cells in the carrier were diluted

with the use of 5mL of 1xPBS The

visualization of the viability of the cells was

performed by the FDAPi staining (fluo-

rescein diacetate and propidium iodide

staining)

Results

The maximum pressure for human (HFF-1)

and mouse (3T3-L1) fibroblasts as well as

for mouse endothelial cells (BALB-5206)

could not be determined for the nozzle 840

μm thus we expected to be above 200 kPa

When we change the diameter to 200μm the

viability of the cells is above 80 when the

pressure is below 190 110 and 170kPa in

the case of HFF-1 3T3-L1 and BALB-

5206 respectively


We confirmed that cells viability is depen-

ded on used pressure and from the inner

diameter of the nozzle used in the bioprin-

ting process We checked 3 cell lines and 5

inner diameters of the nozzle and for each

of the cell lines we designated the maximum

pressure which may be used during bioprin-

ting process and the cell viability wonrsquot fall

below 80

References [1] SV Murphy A Atala Nat Biotechnol vol

32(8) 773-785 2014 doi101038nbt2958

[2] Nair K Gandhi M et al Biotechnol J vol 4(8)

1168-1177 2009 doi101002biot200900004

[3] Frey B Janko C et al Curr Med Chem vol

15(23) 2329-2336 2008 doi10217409298670

8785909166

[4] Blaeser A Duarte Campos DF et al Adv

Healthc Mater vol 5(3) 326-333 2016 doi10

1002adhm201500677

Source of financing

This study was financially supported by The

National Centre for Research and

Development STRATEGMED3305813

2NCBR2017


Chimeric antigen receptor therapies (CAR-T) ndash current knowledge

and perspectives

Krzysztof Kowalski1 Julita Kulbacka

2

1Faculty of Medicine Wroclaw Medical University Poland 2Department of Molecular

and Cellular Biology Faculty of Pharmacy Wroclaw Medical University Poland

147

The joint efforts of biotechnologists and

clinicians in recent years have led to

significant improvements in gene therapies

[1] Despite the initial difficulties associated

with genotoxicity and immune responses

successful clinical trials have enabled the

Food and Drug Administration (FDA) to the

registration of the first treatment in 2014

The dynamic development of CAR-T is

observed in hematological oncology espe-

cially lymphoblastic leukemias This method

involves isolating T-cells from the patients

blood and modifying them ex vivo through a

viral vector mainly lentiviruses and gamma-

retroviruses The main target is to transduce

a gene of the antigen-binding domain

which is fused to an intracellular signaling

domain that mediates activation and co-

stimulation to enhance T cell function and

persistence Recombinant T-cells after

infusion are able to recognize among

others CD19 antigen on cancer cells by-

passing the MHC system and eliminate them

Investigators are intensely focused on better understanding and treating systemic toxicity of therapy because it is still a major prob-

lem Optimizing treatment costs is also a stinging challenge because they are extremely high up to more than 025 mln $ per patient Nowadays researchers are looking for new antigen targets that may allow curing also myeloid malignancies and solid tumors Additionally there is a need for methodologies that facilitate CAR-T cells entry into large tumors and overcome tumor microenvironment signals that disarm T cells

The aim of this project was not only to sum up the latest reports connected CAR-T and present its possibilities in clinical oncology but also to explain the molecular basis of this type of gene editing

Acknowledgments The work was created as part of the activity of the Student Research Group Biology of Cancer Cell at the Wroclaw Medical University (SKN No K 148)

References [1] Dunbar C et al Gene therapy comes of age Science 2018 359


Synthesis proapoptotic and MDR reversal effect of new terpenoid

derivative in colon cancer cells

Agata Kozioła Anna Palko-Łabuz

b Olga Wesołowska

b Krystyna Michalak

b

Mirosława Ferens-Sieczkowska a Kamila Środa-Pomianek

b

aDepartment of Drug Chemistry Wroclaw Medical University ul Borowska 211 50-556 Wroclaw Poland bDepartment of Biophysics Wroclaw Medical University ul Chalubinskiego 10 50-368 Wroclaw Poland both authors contributed equally to this work

Cancer cells often possess intrinsically or develop during treatment the set of features known as multidrug resistance (MDR) The MDR phenotype cells often manifest altered properties such as genome instability or loss of the cell cycle control points which also hinders effective chemotherapy Because of the frequent occurrence of the resistance caused by the

activity of the MDR transporters it is essen-tial to find the effective and ndash at the same time cell-non-toxic ndash inhibitors of those proteins Terpenoid derivatives which contain a preserved β-cyclocitral system in their structure exhibit a broad spectrum of biological activities

Anticancer activity is usually connected with their ability to induce apoptosis In our

148

studies the ability of terpenoid derivative TMPE (3-(266-Trimetylocycloheks-1-en-1-ylo) prop-2-enian ethyl) to induce apoptosis of LoVo HT29 and LoVoDx HT29Dx was confirmed It was also checked whether TMPE could change cytotoxic effect of doxorubicin in sensitive

and resistant sublines In the presence of TMPE cytotoxicity of doxorubicin was elevated and its intracellular accumulation increased In case of LoVoDx and HT29Dx an obtained value of combination index (IC) indicated for a synergistic interaction between doxorubicin and TMPE


Paraoxonase 1 activities in the serum of women with polycystic ovary

syndrome

Maja Krasoń1 Anna Bizoń

2 Grzegorz Franik Paweł Madej

1Faculty of Pharmacy Wroclaw Medical University Poland 2Department of Biomedical and Environmental Analyses Wroclaw Medical University Poland 3Department of Endocrinological Gynaecology Medical University of Silesia Poland

Background

Paraoxonase 1 (PON1) is calcium-depen-

dent enzyme involved in many functions in

human body Various hydrolytic activities

of PON1 can be broadly grouped into three

categories namely arylesterase phospho-

triesterase and lactonase [1] PON1 is

synthesized by the liver and released into

the serum where is associated with HDL

[2] Changes in PON1 status are observed in

different diseases among others in poly-

cystic ovary syndrome Polycystic ovary

syndrome (PCOS) is an endocrine disorder

afflicting females of 18-44 age This disease

leads to infertility insulin resistance obesity

and cardiovascular diseases Since 2003

criteria in accordance with the consensus of

the European Society of Humane Repro-

duction amp Embriology (ESHRE) and the

American Society of Reproductive Medi-

cine (ASRM) established in Rotterdam

according to which the diagnosis of PCOS

requires the presence of 2 of the following 3

symptoms

1 no or rare periods

2 hyperandrogenism hyperandrogenemia

3 image of polycystic ovaries in ultrasound

[3]

The aim of the study was to assay PON1

activities in the blood of women with

PCOS


The study included 40 women with PCOS

aged between 17 and 39 years old and 24

healthy women aged between 17 and 40

years old The value of body mass index

was similar in both groups and was ranged

between 185 and 25 [kgm2]

The diagnosis of PCOS was formed on the

Rotterdam criteria In both groups smoking

and alcohol abuse were among the exclu-

sion criteria The serum were collected

according to the routine procedure after

overnight fasting and stored frozen until

assays In the group of women with PCOS

the blood specimens were collected during

the follicular phase (within 3 and 5 days of

the menstrual cycle)

Phosphotriesterase activity was determined

with paraoxon as a substrate lactonase

activity of PON1 was determined using

dihydrocoumarin as a substrate and aryles-

terase activity of PON1 was determined

using phenyl acetate as a substrate [4]

Results

In the serum of women with PCOS both

PON1 activities arylesterase (7570plusmn2711

149

Ul) and phosphotriesterase (17362plusmn9905

Ul) were statistically significant lower when

compared to the group of healthy women

(11387plusmn3220 Ul and 20521plusmn8021

respectively) However PON1 lactonase

activity was increased in the serum of

women with PCOS (2172plusmn285 Ul) when

compared to group of healthy women

(1817plusmn295Ul) When we divided women

with PCOS according to BMI value we did

not found any statistically significant dif-

ferences between women with PCOS with

BMIlt25 and ge25 However in the group

of healthy women with BMIge25 lower PON

arylesterase activity (9938plusmn2768 Ul) was

observed when compared to the group of

healthy women with BMIlt25

(12421plusmn3119 Ul)


Changes in PON1 activities in the serum

of women with PCOS can be associated

with metabolic disorders

References [1] Aggarwal G Prajapati R Tripathy RK

Bajaj P Iyengar ARS Sangamwar AT Pande

AH 2016 Toward Understanding the Catalytic

Mechanism of Human Paraoxonase 1 Site-Specific

Mutagenesis at Position 192 PLOS ONE 11

e0147999 [2] Efrat M Aviram M Paraoxonase 1 interactions

with HDL antioxidants and macrophages regulate

atherogenesis ndash a protective role for HDL phos-

pholipids Adv Exp Med Biol 2010660153-66 [3] Rotterdam ESHREASRM-Sponsored PCOS

consensus workshop group Revised 2003

consensus on diagnostic criteria and long-term

health risks related to polycystic ovary syndrome

(PCOS) Hum Reprod Oxf Engl 20041941ndash7

[4] Bizoń A Milnerowicz H The effect of divalent

metal chelators and cadmium on serum

phosphotriesterase lactonase and arylesterase

activities of paraoxonase 1 Environ Toxicol

Pharmacol 2018 Mar5877-83


Is using the iQOS system safe for health

Weronika Kraszkiewicz1 Anna Wrona

1 Magdalena Żak

1

1Students Scientific Society at the Department of Toxicolog Faculty Pharmacy Wroclaw


The purpose of our presentation is to eva-

luate the impact on the human body of

alternative tobacco products We pay

special attention to the new product which

exist on Polish market for 3 years it is the

revolutionary iQOS According to the

survey on public opinion it has the

characteristics of a low-risk product

The incineration temperature of tabacco is

different than temperature of burning in

traditional cigarettes or e-cigarettes In

IQOS system the temperature should not

exceed 350ordmC for regular cigarettes

temperature of burning tobacco is approxi-

mately 800ordmC and in e-cigarettes the

incineration temperature is between 150ordmC

and 180ordmC According to many safety

assessment studies of alternative tobacco

products focuses particularly on additional

substances such as allergenic fragrances

or glycerol in electronic cigarettes or tar and

carbon monoxide in traditional cigarettes

Due to information that the research was

sponsored by the manufacturer their

credibility cannot be confirmed and

compare with the safety of e-cigarettes

which are described in detail in the scientific

literature

In our presentation we present the inde-

pendent research of scientists from San

Francisco published in the British Medical

Journal which compared the effects of

150

IQOS e-cigarettes and traditional cigarettes

on the body by affecting the functions of

endothelium of blood vessels and the impact

on lung cells showing their high toxicity In

addition studies by other scientists show

toxic inhibitory effects on macrophages

comparable to traditional cigarettes High

levels of lactate dehydrogenase interleukin

8 in airway epithelial cells and airway

smooth muscle cells have also been

detected which may indicate pathological

conditions

Therefore it seems interesting to examine

public awareness of the risks associated

with the use of the IQOS system As our

own work we want to present the results of

surveys conducted among potential users of

the alternative IQOS system and learn about

their knowledge about this product

References [1] IQOS exposure impairs human airway cell

homeostasis direct comparison with traditional

cigarette and e-cigarette ERJ Open Res 2019 5

00159-2018

[2] Heated tobacco products the example of IQOS

Glantz SA Tob Control 201827s1-s6 doi10

1136tobaccocontrol-2018-054601


Copper(II) coordination compound with tolfenamic acid

Marta S Krawczyk Irena Majerz

Faculty of Pharmacy Wroclaw Medical University

Background

Our research is focused on obtaining new

forms of drugs as potential more effective

pharmaceuticals exhibiting higher solu-

bility stability bioavailability and low

toxicity Synthesis of salts and coordination

compounds is one of the method of

improving of weak solubility and biophar-

maceutical properties of drugs

Tolfenamic acid belongs to the fenamic

acids group known as nonsteroidal anti-

inflammatory drugs (NSAIDs) It acts as

cyclooxygenase (COX) inhibitor and pre-

vents formation of prostaglandins Tolfe-

namic acid is known as a cure for migraine

[1 2]


Studied copper(II) coordination compound

was obtained in the reaction of copper(II)

acetate with tolfenamic acid in methanol in

the form of green needle-like crystals The

crystal structure of the compound was

determined using a four-circle single crystal

X-ray diffractometer at the Faculty of

Chemistry at the University of Wroclaw

The theoretical analysis of chemical bonds

and intermolecular interactions was perfor-

med using QTAIM [3] and noncovalent

interaction (NCI) methods [4]

Results

Copper(II) tolfenamate coordination com-

pound crystallizes in P-1 space group in the

form of dimeric molecular compound that is

build up from two copper(II) centres

coordinated by four tolfenamate ions and

two molecules of methanol Each of the

anions is linked to both Cu(II) ions via

carboxylate group acting as a bridge of

copper centres The coordination sphere of

each Cu(II) ion can be approximated by the

octahedron where O atoms from tolfe-

namate ions are located in vertices of the

square The fifth coordination place of

Cu(II) is occupied by a molecule of metha-

nol while the sixth one is directed toward

the second Cu(II) ion from the dimer

(Figure)

In the crystal structure the network of inter-

molecular weak interactions is observed

151


In the literature there are known a few

structures of meclofenamic acid coordi-

nation compounds with copper(II) and other

transition metals [5]

The crystal structure studies and theoretical

analysis of the copper(II) tolfenamate

coordination compound can provide

a significant insight into its properties and

potential influence on organisms

References [1] K V Andersen S Larsen et al J Chem Soc

Perkin Trans 2 (10) 1443-1447 1989

DOI101039 P29890001443

[2] P J Pentikaumlinen P J Neuvonen et al

European Journal of Clinical Pharmacology 19

(5) 359ndash65 DOI101007bf00544587

[3] R F W Bader (1990) Atoms in Molecules

A quantum Theory Oxford University Press

New York

[4] J Contreras-Garciacutea ER Johnson S Keinan R

Chaudret J-P Piquemal DN Beratan W Yang

J Chem Theory Comput 7 625-632 2011

DOI10 1021ct100641a

[5] D Kovala-Demertzi M Staninska et al

J Inorg Biochem 105 1187-1195 2011

DOI 101016j jinorgbio201105025

Figure Crystal structure of [Cu2(C56H44Cl4N4O8)(CH3OH)2]


Cardiomyocytes contractility improvement after treatment

with 5‐phenyloxyphenyl‐5‐aminoalkyl nitrate barbiturate and ML-7

Anna Krzywonos-Zawadzka1 Marta Banaszkiewicz

1 Agnieszka Olejnik

1 Iwona

Bil-Lula1

1Department of Medical Laboratory Diagnostics Division of Clinical Chemistry

and Laboratory Hematology Wroclaw Medical University Wroclaw Poland

Background

Among the main factors contributing to the

pathogenesis of heart during ischemia

reperfusion (IR) injury are an increased

production of ONOOndash and enhanced

activation of MMP-2 [1] It has been shown

that oxidative stress during IR induces

phosphorylation and nitrationnitrosylation

of myocardial contractile proteins such as

MLC1 and MLC2 [2] Phosphorylation and

nitrationnitrosylation of MLCs increase

152

their degradation by MMP-2 which leads to

heart contractile dysfunction [3] It has also

been shown that ONOO- activates MMP-2

indirectly [4]

Previously we shown that administration

of 5‐phenyloxyphenyl‐5‐aminoalkyl nitrate

barbiturate protects heart against IR injury

[5] The aim of this study was to evaluate if

administration of a mixture of subthreshold

doses of myosin light chain kinase inhi-

bitor (ML-7) and MMPs inhibitor 5‐phe-

nyloxyphenyl‐5‐aminoalkyl nitrate barbi-

turate has protective effect on contractility

of IR hearts Also to determine the effect

of this mixture on levels of MMP-2 and

MLC1


Cardioprotective effect of the subthreshold

doses of drug cocktail was tested on isolated

rat hearts by Langendorf method Hearts

extracted from anesthetized male Wistar rats

(300-350 g) were perfused with Krebs-

Henseleit buffer after 25 min of aerobic

stabilization hearts were subjected no-flow

ischemia (20 min) in the presence or

absence of inhibitors mixture (5‐phenylo-

xyphenyl‐5‐aminoalkyl nitrate barbiturate

(01 microM) and ML-7 (05 microM)) followed by

30 min of aerobic reperfusion Next to

hemodynamic parameters (coronary flow

heart rate left ventricular developed

pressure) biochemical markers of IR injury

were measured in a heart tissue and coro-

nary effluents (MMP-2 LDH) The contra-

ctility of cardiomyocytes was measured

using IonOptix Contractility System

(IonOptix Milton MA USA)

Results

Hemodynamic parameters of cardiac

function were significantly reduced in hearts

subjected to IR compared to aerobic

control Administration of the drug cocktail

improves all analyzed parameters (plt005)

An increased activity of MMP-2 was

demonstrated in heartsrsquo homogenates as

well as perfusates after ischemiareperfusion

in comparison to aerobic control The usage

of the mixture significantly decreased the

activity of MMP-2 (in hearts tissue

p=00357 and perfusates p=00075) Cardiac

tissue damage induced by IR was expressed

by the release of LDH into the coronary

effluent (plt00001) The results showed that

administration of the mixture act caradio-

protective and contributes to the impro-

vement of cardiomyocyte contractility

(p=00004)


We have shown that co-administration of

subthreshold doses of myosin light chain

kinase inhibitor (ML-7) and MMPs inhibitor

(5‐phenyloxyphenyl‐5‐aminoalkyl nitrate

barbiturate) improved cardiac mechanical

function Additionally administration a low

doses inhibitors mixture decrease produc-

tion of LDH and prevents IR induced

increase in MMP-2 activity protects MLC1

from degradation and improve cardio-

myocyte contractility


Science Centre grant no UMO‐2016

23BNZ303151

References [1] L Wang X Cheng et al J Huazhong Univ Sci

Technolog Med Sci 35(1)10-15 2012 DOI

number 101007s11596-015-1381-x

[2] A Doroszko D Polewicz et al Basic Res

Cardiol 104(6)669-679 2009 DOI number

101007s00395-009-0035-1

[3] A Doroszko D Polewicz et al Shock

34(6)592-600 2009 DOI number 101097

SHK0b013e3181e14f1d

[4] VJJ Cadete J Sawicka et al FEBS J 279

2444ndash2454 2012 DOI number 101111j1742-

4658201208622x

[5] AKrzywonos-Zawadzka AFranczak et al

J Cell Mol Med 23(4)2836-2848 2019 DOI

number 101111jcmm14191


153

Antitumor efficacy of ultra-short electrical pulses in murine colon

cancer (MC380) in vivo

Julita Kulbacka1 Natalia Anger-Goacutera


1 Joanna Rossowska

2

1 Department of Molecular and Cellular Biology Faculty of Pharmacy Wroclaw Medical

University Poland 2 Hirszfeld Institute of Immunology and Experimental Therapy

Wroclaw Poland Correspondence julitakulbackaumedwrocpl

Background

The application of ultra-short electrical

pulses in cancer treatment is in research

focus during past ten years This technique

is promising for tumor ablation without

usage of cytostatic drugs [1] Very short

electrical pulses (3-600 ns) can be applied

with minimal Joule heating causing insig-

nificant thermal effects in cells (temperature

rise up to 3degC) [2] Extracellular matrix

nerves and vessels are preserved in the

absence of heating what promotes quick

repopulation of the treated area with normal

cells and fast recovery of treated tissues

Among other methods nsPEF ablation is

quite a new methodology and is subject for

intensive study

The aim of this study was the application of

ultrashort pulses (10 ns) and high intensities

of electric fields as the form of anti-tumor

therapy in murine model of colon

carcinoma


Murine colon carcinoma cells (MC380)

were inoculated subcutaneously (sc) into

right flank of C57BL6 mice When tumors

reached a volume of 50 mm3 nsPEF proto-

cols were applied We have applied 10 ns

pulses with following parameters of nsPEF

125kVcm and 400 or 1200 pulses and

25kVcm and 400 or 1200 pulses After

treatment the MC380 tumor growth inhi-

ition was evaluated

Results

The obtained results indicate the propor-

tional decrease of tumor volume with the

increasing electroporation parameters The

highest anticancer potency was obtained for

25kVcm and 1200 pulses


Ultra-short electrical pulses seems to be

a promising and low invasive type of anti-

cancer therapy Similar studies were perfor-

med by Novickij et al [3] where also 12

kVcm electric field intensity was applied

but longer 200ns pulses Authors used

myeloma tumor models and also observed

decreasing tumour volume with the incre-

asing parameters of PEF Thus we can state

that nanosecond irreversible electroporation

can induce an anti-tumor response

Funding

The research was supported by National

Science Centre (Poland) within a framework

of SONATA BIS 6 (201622ENZ500671

PI J Kulbacka)

References [1] M Breton and L M Mir Bioelectromagnetics

vol 33 no 2 pp 106-123 Feb 2012 doi

101002bem20692

[2] K H Schoenbach S J Beebe et al

Bioelectromagnetics vol 22 no 6 pp 440-448

Sep 2001 doi 101002bem71

[3] V Novickij et al Cancers (Basel) vol 11 no

11 Nov 2019 doi 103390cancers11111763


154

Effectiveness and toxicity of supplements containing red yeast rice

and Monacolin K

Wiktoria Kurzyna1 Paulina Matyśkiewicz

1 Aneta Ligenza

1 Anna Milczanowska

1

Ewa Sawicka2


Medical University Wroclaw Poland 2Department of Toxicology Faculty Pharmacy


Background

Red yeast rice (RYR) is a food product used

in the traditional Chinese cuisine It is made

from Oryza Sativa under the influence of

Monascus yeast The fermentation process

enriches the rice with a compound that has

a mechanism of action similar to that of

statins ndash Monacolin K Despite the identical

structure the therapeutic effect of Monacolin

K differs from the effect caused by

lovastatin Differences in bioavailability and

pharmacokinetics of those substances occur

what most probably results from the actions

of other chemical compounds that are

present in the RYR Increase in popularity

of dietary supplements containing an extract

from red yeast rice can be observed within

the recent years The substance is recom-

mended as an adjuvant in the treatment of

hypercholesterolemia Red yeast rice is

specially valued in the case of patients

allergic to synthetic statins


Our poster shows various clinical trials

focused on Monacolin K and its effects on

health

Results

The cholesterol-lowering effect of red yeast

rice has been confirmed in several meta-

analyses of randomized controlled clinical

trials One of the trials proved that the

intake of the substance within the time from

2 to 24 months reduced LDL-C by an ave-

rage of 102 mmolL compared to placebo

what is a similar effect as that of moderate-

intensify statins

Discussions and conclusions

However some doubts concerning safe use

of those substances occur Monacolin is

being sold as dietary supplements with less

strict legal requirements What is more red

yeast rice might be contaminated with

citrinin ndash a nephrotoxic and hepatotoxic

substance Moreover its teratogenic effects

has also been reported

Monacolin K influences on the activity of

liver enzymes what may lead to changes in

metabolism of different drugs such as

ciclosporin fibrates macrolides and vera-

pamil In the cases of particularly vulnerable

patients monacolin K might cause

myopathy and rhabdomyolysis Due to the

two faces of Monocalin K its supple-

mentation must be controlled and and its

toxic properties described in detail to avoid

intoxication

References [1] Cicero AFG Fogacci F (2019) Red Yeast Rice

for Hypercholesterolemia Methodist Debakey

Cardiovasc J15(3)192-199

[2] Gerards Maaike C et al (2015) Traditional

Chinese lipid-lowering agent red yeast rice results

in significant LDL reduction but safety is uncertain

ndash A systematic rewiev and meta-analysis

Atherosclerosis Volume 240 Issue 2 415-423


155

Steroid receptor RNA activator 1 in arteries

Aleksandra Kuzan1 Karolina Nowakowska

1 Agata Kotowska Anna Stebnicka

1 Faculty of Medicine Wrocław Medical University

Background

Steroid receptor RNA activator 1 (SRA1)

is considered to be long noncoding RNA

(lncRNA) [1] and functional RNA encoding

the protein ndash Steroid Receptor RNA

Activator Protein (SRAP) [2] Both coding

and non‐coding SRA transcripts co‐exist in

human cells and acts as transcriptional

regulator multiple nuclear receptors nuclear

receptor co‐regulators and protein involved

in gene silencing [3]

SRA1 is attributed to roles in regulating

cellular response to estrogen and androgen

stimulus glucose uptake the process of

apoptosis cell differentiation and proli-

feration thereby affecting steroidogenesis

myogenesis tumorigenesis and cardio-

myopathy [34]

It is reported that SRA1 is highly expressed

in liver skeletal muscle adrenal gland and

the pituitary gland intermediate expression

levels are observed in the placenta lung

kidney and pancreas in the brain and other

typical steroid-responsive tissues such as the

prostate breast uterus and ovary the expres-

sion is rather low [5] This protein is also

found in case of several human tumors

types mainly tumours of the breast liver

and bone [4] The reports describe the high

expression in smooth muscle but they does

not specify whether it is vascular smooth

muscle or for example the digestive system

muscles [6] No information has been found

in the literature on the subject whether

SRA1 is present in the aorta and whether

it has any significance in the pathome-

chanism of atherosclerosis Our hypothesis

is that it can control the migration of

myocytes from the intima to the media and

the proliferation of these cells contributing

to development of atherosclerotic plaque


The quality and semi-quantitative analysis

of SRA1 content in 27 sections of the

human aorta which are at various degrees

of atherosclerosis (according to the Ame-

rican Heart Association) was performed

This study was approved in terms of ethics

by the Bioethics Committee of Wroclaw

Medical University (no 5772017) The

samples came from people who died

a sudden death (average 68plusmn15 years old)

The standard immunohistochemical method

was carried using anti SR-A1 rabbit mono-

clonal antibodies conjugated with HRP

(1100 Abcam ab202922)

Results

The SRA protein is particularly visible in

the area of the fatty core in the region of

cholesterol fissures formation The antigen

is visible in point form In some patients the

antigen also occurs in myocytes of media

altered by atherosclerosis It can also be

found in adventitious cells Only trace

amounts of antigen are observed in probes

at low atherosclerosis level


Up to date it is not possible to precisely

define the function of SRA1 In the context

of the impact of SRA1 on cell migration and

proliferation we have data showing that

SRA can inhibit the proliferation of certain

cells [4] but can also be attributed to the

growth of others [7] Reduced expression in

some types of cancers [8] also suggests that

this protein rather inhibits proliferation

We discover a tendency to increase protein

expression in atherosclerotic plaque This

result suggests that SRA1 may contribute to

the stimulation of migration and or proli-

156

feration of myocytes in the wall of large

blood vessels such as the aorta

As future direction we plan to examine

content of noncoding and coding transcripts

of SRA1 in the tissue

References [1] C Liu H T Wu et al Clin Chim Acta vol

459137-146 2016 doi 101016jcca201606004

[2] S Chooniedass-Kothari E Emberley et al

FEBS Lett vol 56643-47 2004 DOI 101016

jfebslet200403104

[3] Y Yan C Cooper et al FEBS Lett vol 589(24

Pt B)4010-8 2015 DOI 101016jfebslet

201511007

[4] W Guo H Jiang et al Technol Cancer Res

Treat vol 181533033819841438 2019 DOI

1011771533033819841438

[5] E Leygue Nucl Recept Signal35e006 2007

DOI 101621nrs05006

[6] httpwwwproteinatlasorgENSG00000

213523-SRA1tissue

[7] K Lin H Zhan et al Reprod Sci vol

24(6)836-843 2017 DOI 101177193371911

6670036

[8] P Luo W Jing et al Cancer Biomark vol

18(3)285-290 2017 DOI 103233CBM-160305


Voltammetric determination of fenamic acid on poly-N-acetylaniline

modified glassy carbon electrode

Anna Kwiecień1 Adam Sroka

1 Irena Majerz

1

1Faculty of Pharmacy Wroclaw Medical University

Background

Fenamic acid (FA) is a derivative of anthra-

nilic acid The compound is a parent struc-

ture for several non-steroidal anti-inflam-

matory drugs mefenamic acid tolfenamic

acid flufenamic acid and meclofenamic

acid [1] Due to its side effects fenamic acid

is not used in pharmacy [2]

Voltammetry is a method characterized by

high sensitivity accuracy precision and

broad linearity range with relatively low-

cost apparatus [3] This technique was

successfully used for the determination and

assay of many drugs [4] Chemically modi-

fied electrodes (CMErsquos) are quite new

approach in basic electrochemical experi-

ments The deposition of thin polymer film

on the electrode surface endow the CME

with desirable chemical or electrochemical

properties

In the current work poly-N-acetylaniline

(PNAANI) glassy carbon (GCE) modified

electrode was used for determination of

fenamic acid in aqueous media


Electrochemical measurements were

performed on a multipurpose Electroche-

mical Analyzer M161 with the electrode

stand M164 (both MTM-ANKO Poland)

A three-electrode single-compartment cell

was used for cyclic voltammetry A chemi-

cally modified electrode was used as the

working electrode a platinum wire as the

counter electrode and a AgAgCl electrode

as the reference electrode The working

electrode was prepared by electrodeposition

of poly-N-acetylaniline film on the surface

of GCE A 20mM stock standard solution

of sodium fenamate was prepared A 8-ml

solution containing an appropriate amount

of fenamic acid and 03M Britton-Robinson

buffer solution was transferred into

the voltammetric cell Cyclic voltammetry

(CV) experiment was conducted by

potential sweeping from -250 to 1000 mV

Results

The effect of pH on fenamic acid oxidation

was investigated in the pH range from 174

157

to 651 The peak current and peak potential

are dependent on pH of Britton-Robinson

buffer The pH value of 330 was chosen as

the supporting electrolyte The relationship

between the Imax for anodic peak current and

the concentration of fenamic acid was

examined by CV in the pH 330 in Britton-

Robinson buffer at the scan rate of 100

mVs (Figure 1)

bull Anodic peak current increased linearly

with the FA concentrations from 625 times 10minus7

to 300 times 10minus5 molL

bull A detection limit of 618 times 10minus11 molL

was obtained


The oxidative reaction of fenamic acid can

be used as an assay of the compound in the

linear range of concentration 0625μM to

30μM with detection limit of 618pM Liner

range is limited to 30μM by the low

solubility of fenamic acid the supporting

electrolyte

References [1] GG Graham Fenamates in Compendium

of Inflammatory Diseases (Parnham M J Ed)

477-482 Springer Basel 2016 Basel

DOI 101007978-3-0348-0620-6_24-1

[2] TL Hardy PH Bach Toxicology and Applied

Pharmacology vol 75 265-277 1984

DOI 1010160041-008X(84)90209-6

[3] SA Ozkan B Uslu Journal of Pharmaceutical

and Biomedical Analysis vol 130 126-140 2016

DOI 101016jjpba201605006

[4] N Abo El-Maali Bioelectrochemistry vol 64

99-107 2004 DOI 101016jbioelechem2004

03003

Figure 1 Calibration curve


158

Preparation of nesfatin-1 ndash a novel multifunctional hormone peptide

Rafał Lenda1 Dominika Bystranowska



Background

Human nesfatin-1 is a 82-amino acid

peptide hormone that was first discovered

by Oh-I et al [1] Nesfatin-1 is mainly

involved in regulation of energy homeo-

stasis and exhibits potent anorexigenic

effect independent of leptin signalling when

injected intracerebroventricularly in mice

[2] Nonetheless this hormone may be

involved in many other important biological

processes such as regulation of anxiety and

stress [3] reproduction [4] regulation

of circadian rhythm and epilepsy [56]

Recent studies show that high levels of

nesfatin-1 may be correlated with metastasis

and poor prognosis of colorectal cancer [7]

bladder cancer [8] or breast cancer [9]

Development of effective preparation

methods of nesfatin-1 is thus important

Herein we present an efficient protocol for

preparation of recombinant human nesfatin-

1 from E coli cells


Buffers A (300 mM NaCl 50 mM

Na2HPO4 pH 70) B (buffer A supple-

mented with 35 mM imidazole pH 70) C

(buffer A supplemented with 200 mM

imidazole pH 70) D (150 mM NaCl 20

mM Tris-HCl pH 75) All agents were

purchased from Roth beside NaCl which

was purchased from Merck

Affinity chromatography Bacteria were

lysed by sonication (85s bursts 65s pause

40 intervals) and 50 μl of DNase (10 mgml

Sigma) and RNase (10 mgml Sigma) were

added Then the extract was centrifuged

(12 000 timesG 4degC 50 min) and supernatant

was collected From then the experiment

was conducted at 4 degC Next 1 ml of Ni-

NTA HisbullBindreg Resin (Merck) was equili-

brated with 10 ml of buffer A and then

incubated with the supernatant on the

vertical shaker (12 RPM 30 min) Super-

natant was then loaded onto the column and

the flow-through (FT1) was collected Next

the column was washed with 10 ml of

buffer A and the wash fraction was collec-

ted The column was rinsed with 5 ml of

buffer B and 05 ml fractions were

collected Finally nesfatin-1 was eluted with

5 ml of buffer C and 05 ml fractions (E1-

E10) were collected Concentration of

nesfatin-1 was estimated by measuring

A280 (Nanodrop 2000c Thermo Scientific)

HRV3C-tag digestion and nesfatin-1

purification Fractions E2-E6 were pooled

together and desalted to buffer A on the

PD10 column (GE Healthcare) according to

manufacturerrsquos instructions Next the

concentration of nestatin-1 was estimated

with A280 and the excitation coefficient of

045 mltimesmg-1timescm-1 and HRV3C (Sino

Biological) protease was added in the ww

ratio of 1100 to remove the His-tag The

digestion was carried out on vertical shaker

(12 RPM 4degC 12 h) Subsequently 200 μl

of Ni-NTA resin was equilibrated with 4 ml

of buffer A and incubated with the digestion

solution on the vertical shaker (12 RPM

4degC 30 min) Then the solution was loaded

onto the column and flow-through (FT2)

was collected Next FT2 was concentrated

on the Amicon Ultra-4 (Merck) to about 500

μl volume On each step the concertation of

nesfatin-1 was estimated based on A280

measurement

Size-exclusion chromatography Further purification of nesfatin-1 was carried out on the Superdex 75 Increase 10300 (GE

159

Healthcare) column First the column was equilibrated with buffer D and then the sample was injected The fractions exhi-biting nesfatin-1 presence were pooled and stored at -80degC until further evaluation

Results First stage of affinity chromatography yields about 2 mg of nesfatin-1 per 05 L of bacterial culture At this stage the protein is slightly contaminated with bacterial proteins as proved by SDS-PAGE and Western-blot analysis The subsequent steps yield about 1 mg of pure and homogenic nesfatin-1 sample as evidenced by SDS-PAGE Western-blot and mass spectrometry analysis

Discussion and conclusions The protocol presented here allows for fast and efficient preparation of human nesfatin-1 which to our knowledge hasnrsquot been described in the literature yet We hope that

this protocol will endow further research on this unique peptide

References [1] S Oh-I H Shimizu et al Nature 2006 443 (7112) 709-712 DOI 101038nature05162 [2] H Shimizu S Oh-I et al Endocrinology 2009 150 (2) 662-671 DOI 101210en2008-0598 [3] Z Merali C Cayer et al Psychopharmacology 2008 201 (1) 115-123 DOI 101007s00213-008-1252-2 [4] D Garcia-Galiano V M Navarro et al Journal of Neuroscience 2010 30 (23) 7783-7792 DOI 101523JNEUROSCI5828-092010 [5] A Pałasz M Krzystanek et al Neuropeptides 2012 46 (3) 105-112 DOI 101016jnpep 201112002 [6] S Aydin E Dag et al Molecular and Cellular Biochemistry 2009 328 (1ndash2) 49-56 DOI 101007s11010-009-0073-x [7] J Xie L Chen et al Oncology Letters 2018 15 (6) 9188-9194 DOI 103892ol20188523 [8] G M Liu Z Q Xu et al Disease Markers 2018 2018 1-9 DOI 10115520184272064 [9] L Zeng J Zhong et al Journal of Cancer 2017 8 (15) 3062-3069 DOI 107150jca19619


Correlations between selected markers associated with the red-ox

status with selected parameters of inflammation and glycaemic status

in a mixed obese-non-obese population sample


2 Marta Kepinska


1

1Department of Biomedical and Environmental Analyses Faculty of Pharmacy Wrocław Medical University 2Department of Medical Laboratory Diagnostics Faculty of Pharmacy Wrocław Medical University

Background As inflammation and increased body fat are coexistent with changes in the total antioxi-dative capacity (TAC) much research into antioxidants has been carried out in order to determine the differences between obese and non-obese individuals and associated alterations in basic clinical parameters describing glycaemic status ongoing inflammation process and insulin resistance

In this research monotonic correlations between clinical parameters and selected markers associated with the red-ox status in

plasmaserum were checked for The main purpose was to assess possible associations between the patterns in which oxidative stress affects antioxidative parameters mainly ndash the concentration andor activity of superoxide dismutase isozymes

Material and Methods The sample consisted of 94 individuals of which 50 were non-obese and 40 ndash obese as determined by a BMI cutoff (BMI ge 30) The male-to-female ratio in both non-obese and obese was moreless equal (2129 and 2420 accordingly)

160

The variables between which the correla-tions were tested are concentration of superoxide dismutase isozymes (SODs) SOD1 SOD2 SOD3 total SOD activity CuZn-SOD activity Mn-SOD activity TAC concentration of malondialdehyde (MDA) copper and zinc cadmium C-reactive protein (CRP) glucose and insulin concentration HOMA-IR (insulin resistance parameter) age and BMI

Results The power of most of the significant correlations in this study was below ρ 05 Age negatively correlated with the activity of CuZn-SOD TAC concentration of MDA copper and cadmium CRP nega-tively correlated with total SOD activity positively ndash with TAC MDA and copper concentration Glucose concentration correlated positively with TAC MDA and zinc concentration whereas insulin concen-tration correlated negatively with total SOD activity and positively with SOD1 concentration TAC and MDA HOMA-IR correlated with the same factors as insulin except for MDA BMI correlated negatively with total SOD activity CuZn-SOD activity and positively with TAC MDA and cadmium

Interestingly the concentration of SOD1 weakly and negatively correlated with the concentration of SOD2 but positively with CuZn-SOD activity TAC and zinc concentration Both total SOD and Mn-SOD activity correlated negatively with copper concentration TAC correlated with MDA

Discussion and conclusions No strong associations have been found between any of the described variables The association between the concentration of zinc and the concentration of SOD1 and TAC might be due to the fact that zinc is an antioxidant present in the active site of SOD1[1] Decrease in SOD activity with an increase in age has been previously shown [2]

The positive correlations between TAC and BMI HOMA-IR and CRP glucose insulin and MDA concentration may indicate that TAC is upregulated in strongly prooxidative conditions presumably as means of adap-tation aimed to restore red-ox homeostasis The possibility of the organism to adapt to oxidative stress by upregulation of antioxi-dative capacity has been previously shown in skeletal muscles of non-insulin dependent type 2 diabetic men [3] Negative correla-tions between CRP insulin HOMA-IR and BMI with total SOD activity may indicate inactivation caused by oxidative modify-cations of SOD isozymes or other processes ongoing processes such as glycation [4 5] The lack of association between any of superoxide dismutase activities and TAC seems to support the thesis stating that TAC assays mostly measure antioxidative capa-city of low molecular mass compounds [6]

The correlations found in this study seem logical However there are discrepancies between the correlations found in this study and these found in the literature In a study by Lim et al BMI correlated negatively with TAC [7]

References [1] JJP Perry DS Shin et al Biochim Biophys Acta vol 1804 2 2010 DOI 101016jbbapap 200911004 [2] PK Maurya P Kumar et al Ind J Biochem Biophys vol 47 2010 [3] Brinkmann C Chung N et al Scand J Med Sci Sports vol 22 4 2012 DOI 101111j1600-0838201001273x [4] N Kawamura T Ookawara et al J Clin Endocrinol Metab vol 74 6 1992 DOI 101210jcem7461592880 [5] A Oda C Bannai et al Horm Metab Res vol 26 01 1994 DOI 101055s-2007-1000762 [6] Young IS J Clin Pathol vol 54 5 2001 DOI 101136jcp545339 [7] SH Lim SH Fan et al Mal J Nutr vol 18 3 2012


161

The role of apoA-I in regulation of paraoxonase-1 (PON1) activity

Dominika Lewoń1 Milena Ściskalska


2

1Students Scientific Association at Department of Biomedical and Environmental Analyses Wroclaw Medical University Faculty of Pharmacy Wroclaw Poland 2Department of Biomedical and Environmental Analyses Wroclaw Medical University Faculty of Pharmacy Wroclaw Poland

Background PON1 is a Ca2+-dependent glycoprotein with a molecular mass of 43 kDa PON1 are primary synthesized in the liver and then secreted into the bloodstream where it circulates throughout the body in asso-ciation with high density lipoproteins (HDL) particles [1] PON1 can be evaluated by its different activities paraxonase activity (against organophosphates such as paraoxon) arylesterase activity (against aromatic esters such as phenylacetate) and lactonase activity as a native activity of PON1 (against lactone-like structures elabo-rated by oxidized polyunsaturated fatty acids on lipoproteins) [234] The role of PON1 is the protection from the oxidation of HDL and LDL molecules This enzyme by destroying oxidized phospholipids reduces the ability of oxidized LDL to induce monocyte chemotaxis and thus limit the inflammatory processes in the vessel wall and inhibit the development of vascular and coronary diseases [1]

The antioxidative function is provided by the presence of HDL molecule in PON1 structure which contains apoliprotein AI (apoAI) [5] Aim of the paper was to analyze studies investigating influence of lipoprotein ApoA-I on PON1 activity

Material and Methods Data was collected by anlyzing available articles which present results of studies related to PON1 and its association with apoA-I Data were sought by computer-based searches from databases including PubMed Google Scholar without language restriction Search term combinations were keywords relating to the paraoxonase 1

(eg bdquoparaoxonaserdquo bdquoPONrdquo bdquoPON1rdquo bdquoPON1 activityrdquo bdquoPON1 activity regu-lationrdquo) in combination with words related to apoA-I (eg bdquoapoA-Irdquo bdquoPON1 and apoA-Irdquo) One term was replaced each time until all possible combination mode were searched to avoid any missing literature The titles and abstracts of potential articles were screened to determine their relevance Chosen literature represent researches conducted between 1999 and 2019

Results

A number of studies have demonstrated that HDL provides a vector that facilitates the secretion of the enzyme by liver essentially by offering a hydrophobic harbor for the retained signal peptide of PON1 and coincidentally stabilizing the enzyme The lipoprotein such as apoA-I also furnishes a hydrophobic environment that is important for PON1 function [6] A positive correla-tion between apoA-I and PON1 levels in vivo combined with its absent or low activities observed in apoA-I deficiencies (such as Tangier disease) suggested that apoA-I was required for the expression of PON1 as well as its binding to HDL [7] It was shown positive associations between PON1 activitiesconcentrations and HDL-cholesterol and apolipoprotein A-I (apoA-I) concentrations [6] Oda et al revealed that apoA-I increases PON activity in a concen-tration-dependent manner however it doesnrsquot influence PON1 secretion from cells [8] Sorenson et al were noted that apoA-I is not required for the association of PON1 with phospholipid-containing vesicles however serum PON1 activity is more stable in the presence of apoA-I and if

162

apoA-I is absent PON1 activity decreases rapidly [5 7]

Discussion and conclusions Serum concentration of lipoprotein apoA-I might affect activity of PON1 Although PON1 binding to lipoproteins does not require apoAI its presence is necessary to maintain optimum activity and stability of the enzyme [8]

References [1] Y Zhao Y Ma et al Molecular Genetics and Metabolism vol 105 141-148 2012 DOI 101016jymgme201109018 [2] J Ceron F Tecles et al BMC Veterinary Research vol 10 74 2014 DOI 1011861746-6148-10-74

[3] J Corsetti Ch Sparks et al J Clin Med vol 8 1357 2019 DOI 103390jcm8091357 [4] D Levy C O Reichert et al Antioxid Basel Switz vol 8 2019 DOI doi 103390 antiox8050118 [5] S Deakin R James Clinical Science vol 107 435-447 2004 DOI 101042CS20040187 [6] M Blatter Garin X Moren et al Journal of Lipid Research vol 47 2006 DOI 101194 jlrM500281-JLR200 [7] R Sorenson C Bisgaier et al Arterioscler Thromb Vasc Biol vol 19 2214-2225 1999 DOI 192214-2225

[8] M Oda J Bielicki et al Biochemistry vol 40

1710-1718 2001 DOI 101021bi001922h


Xenobiotics and estrogens as potential inducers of lipotoxicity

Katarzyna Lipke1 Agnieszka Piwowar

2

1Student of Faculty of Pharmacy Wroclaw Medical University 2Department of Toxicology


Adipose tissue is not only responsible for

storage of energy in form of lipids in human

body (white adipose tissue) or generating

body heat (brown adipose tissue) but also

has been recognised in recent years as an

endocrine gland [1] Hormones produced by

adipocytes are referred to as adipokines and

play vital role in regulating fatty acids (FA)

(in form of triacylglicerol (TG)) reservoirs

in a cell [1 2] Main storage of FA is

located in adipose tissue but also a small

reservoir of FA is present in almost every

human cell because FA are needed for

example in process of phospholipid bilayers

formation [2] Adipokines are responsible

for proper distribution of FA in body cells

in cases of normal nutrition overnutrition

and malnutrition When the homeostasis of

adipokines is disrupted or when the adipose

cells become unresponsive to adipokines

FA start to cumulate in nonadipose cells in

larger amounts than they should resulting in

general steatosis which leads to disfunction

of nonadipose tissues [2] Originally this

state was described as lipotoxicty but

currently it is known as a more complex

state Too high amount of FA has destruc-

tive effects on glucose metabolism and it

causes functional impairments in several

metabolic pathways both in adipose tissue

and peripheral organs like liver heart and

muscle Additionally lipotoxicity plays an

important role in insulin resistance and

pancreatic beta cell dysfunction [3]

There are many potential factors both endo-

and exogenous that can disturb organism

hormonal balance and thus may induce

lipotoxicity Different xenoestrogens as an

exogenous factors or estrogens and its

metabolites as an endogenous agents both

are indicted as endocrine disruptors and

potential inducers of lipotoxicity A syste-

matic review of the literature data relating

to lipotoxicity in the aspect of xenobiotics

and estrogens action was performed

163

Estrogens and its metabolites act mainly by

estrogen receptors inducing DNA destruc-

tion intracellular signaling pathway changes

andor oxidative stress [4] These pathways

can promote cells disturbances and they can

also indirectly induce intracellular endocrine

disturbances and probably promote the

development of lipotoxicity Xenobiotics

may affect the level of adipokines in blood

serum adipocytesrsquo response for adipokines

leading to change of FA concentratios

therefore to insulin resistance early stages

of diabetes and steatosis of tissues More-

over the metabolism of FA (usually on the

oxidative way) takes alternate nonoxidative

pathway which results in the rise of TG

content Products of further metabolism

of TG may lead to dysfunction and even

death of cells [2] Free FA may also induce

endoplasmic reticulum and mitochondrial

stress [56] Additionally xenoestrogens can

mimic the action of estrogen causing

changes in cells similar to those induced by

estrogens One of the representatives of

xenobiotics group with the potential

of influencing hormonal homeostasis can

be metalloestrogens such as cadmium

nickel antimony which are factors that can

exacerbate lipotoxic condition [7 8]

Investigation of phenomena of lipotoxicity

caused by xenoestrogens and estrogens may

elucidate yet unknown mechanisms of

endocrine disruption connected especially

with visceral adiposity and insulin resis-

tance which increase the cardiometabolic

risk and risk of metabolic syndrome deve-

lopment Lipotoxicity seems to play

a crucial role in the pathophysiology of

these complex associations Researching

and understanding the possible mechanisms

of interaction between the xenobiotics and

estrogens mentioned above will provide

valuable scientific information and guidance

on the preventive actions of adverse effects

of lipotoxicity

References [1] H Tilg A R Hoschen Nature vol 6 772-783

2006 DOI101038nri1937

[2] R H Unger Annu Rev Med Vol 53319ndash36

2002 DOI 101146annurevmed53082901

104057

[3] D Yazici H Sezer Adv Exp Med Biol Vol

960 277-304 2017 doi 101007978-3-319-

48382-5_12

[4] E Sawicka A Woźniak et al Postepy Hig Med

Dosw Vol 73 909-919 2019 DOI105604

01300100137541

[5] D A Cunha P Hekerman et al Journal of Cell

Science Vol 121 2308-2318 2008 DOI10

1242jcs026062

[6] T J Biden E Boslem et al Trends in

Endocrinology and Metabolism Vol 25 389-

398 2014 DOIhttpsdoiorg101016jtem2014

02003

[7] A Stoica B S Katzenellenbogen et al

Molecular Endocrinology Vol 14 545-553 2000

httpsdoiorg101210mend1440441

[8] S-Y Choe S-J Kim et al The Science of the

Total Environment Vol 312 15-21 2003

doi101016S0048-9697(03)00190-6


A different strategy to prevent protein adsorption

Dawid Lupa1

1Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences

Background

The nonspecific adsorption of proteins at

various interfaces is well-known undesi-

rable phenomenon that could possibly lead

to deterioration of the medical implants [1]

Given that nonspecific adsorption of

proteins must be suppressed different

strategies for chemical modification of

surface properties of the implants are

constantly developed These strategies can

be divided into three main categories cova-

164

lent grafting of polymer brushes surface

initiated polymerization and chemisorption

of thiol-terminated polymers Despite the

effort devoted to develop aforementioned

strategies their application is still limited

mainly due to the need of harsh chemical

usage For this reason in this work we

present an alternative approach consisting of

immobilization of poly(styreneα-tert-

butoxy-ω-vinylbenzylpolyglycidol)

microparticles ndash P(SPGly) in diffusion

controlled electrostatic-driven process


Polymer microparticles were synthesized in

radical surfactant-free emulsion polymeri-

zation in aqueous medium After purify-

cation the basic physicochemical properties

of investigated microparticles such as hydro-

dynamic diameter and zeta potential were

determined for broad range of pH using

Malvern Zetasizer apparatus Silicon plates

modified by adsorption of poly(allylamine

hydrochloride) were chosen as model

surface utilized for streaming potential and

adsorption kinetics studies NT-MDT Solver

Atomic Force Microscope (AFM) were

used to determine both roughness of silicon

plates and adsorption kinetics of micro-

particles Antifouling properties of obtained

surfaces were determined using streaming

potential measurements In this case

a monolayer of microparticles was obtained

directly in the streaming potential cell and

streaming current was determined After-

ward the cell was flushed and filled with

human serum albumin (HSA) solution HSA

was adsorbed under diffusion controlled

conditions for certain amount of time

Afterward the cell was flushed with NaCl

solution and streaming potential was

determined again

Results

Firstly the hydrodynamic diameter and zeta

potential of microparticles were determined

for pH range 4-10 and ionic strength equal

to 10-2 M and 10-3 M It was found that

hydrodynamic diameter of microparticles is

equal to 350 nm for both investigated ionic

strengths Moreover it was found that

hydrodynamic diameter does not depend on

pH value This is in agreement with diame-

ters derived from SEM analysis Performed

zeta potential measurements proved that

P(SPGly) microparticles exhibit negative

surface charge in whole investigated pH

range To be more precise the zeta potential

determined for ionic strength equal to 10-2

M diminished from -45 mV to -60 mV

when pH changed from 4 to 10 This confir-

med the possibility of P(SPGly) micro-

particles immobilization on positively

charged surface of PAH-modified silica

From adsorption kinetics experiments perfor-

med for different adsorption time (1-40 h) it

was found that experimentally determined

adsorption rate is twofold lower compared

to the theoretical one Moreover it was

revealed that jamming coverage of investi-

gated microparticles is significantly higher

than maximum coverage predicted by RSA

model for spherical rigid spheres Finally

HSA adsorption measurements revealed that

compared to bare PAHSiO2 surface the

amount of adsorbed HSA is threefold

smaller for PAHSiO2 surface modified by

adsorption of P(SPGly) microparticles


Kinetic measurements performed for

P(SPGly) microparticles immobilization at

PAHSiO2 surface enabled to determine the

structure of investigated microparticles

It was found that effective density of such

particles was equal to 06 g ml-1 which is

considerably lower that density of typical

polystyrene microparticle (105 g ml-1) This

results from the presence of polyglicidol-

rich fuzzy shell layer which thickness was

estimated to be equal to 25 nm The struc-

ture of investigated particles was confirmed

both by streaming potential and AFM

measurements Streaming potential method

165

enabled also a determination of anti-fouling

properties of particle monolayers It was

confirmed that for modified surfaces the

adsorption of HSA was noticeably lower

(04 mg m-2) than in the case of bare one

(13 mg m-2) [2] This effect can be attri-

buted to the presence of fuzzy shell layer

with properties similar to polymer brush In

such case the proteinsurface interaction

energy is significantly lower due to preven-

tion of HSA molecules from approaching

the surface and thus forming a strong

electrostatic-driven interaction One can

therefore expect that immobilization of

P(SPGly) microparticles can be efficient

strategy for creating controlled protein-

repelling surfaces

References [1] Y Hedberg Materials Degradation vol 2 26

2018 doi101038s41529-018-0049-y

[2] M Wasilewska Z Adamczyk et al Langmuir

vol 32 9566-9574 2016 DOI101021acs

langmuir6b02069


Does p53 mediate the upregulation of selected genes induced by cell

treatment with actinomycin D and nutlin-3a

Barbara Łasut-Szyszka1 Małgorzata Krześniak

1 Agnieszka Gdowicz-Kłosok

1

Artur Zajkowicz1 Iwona Matuszczyk

1 and Marek Rusin

1

1Center for Translational Research and Molecular Biology of Cancer Maria Skłodowska-

Curie Institute ndash Oncology Center Gliwice Branch Poland barbaralasutiogliwicepl

Background

Fourty years ago p53 was discovered as one

of the first tumour suppressor genes Studies

carried out all over the world showed the

multifaceted role of p53 in regulation of the

response to cellular stress such as DNA

damage hypoxia oncogen activation or

viral infection Despite intensive research on

TP53 gene and its protein there are still

unidentified mechanisms concerning

functioning of p53

We have observed that two substances

which stimulate p53 in different ways

actinomycin D and nutlin-3a when acting

simultaneously (A+N) induce synergistic

activation of p53 in different cancer cell

lines and in normal human fibroblasts

Probably the synergy of these molecules

results from the fact that actinomycin D

stimulates phosphorylating of p53 by

various kinases whereas nutlin-3a helps the

kinases in efficient phosphorylation of p53

by blocking the negative regulator of p53

the MDM2 protein The analysis of

transcriptome sequencing (RNA-Seq) of

cancer cell lines exposed to A+N has

revealed a significant increase in the

expression of over 2000 genes including

expression of 500 genes up-regulated at

least 10-fold Based on our results and

available binding site databases we have

found several genes not yet associated with

p53 regulation genes negatively regulating

signalling through Wnt pathway DRAXIN

FRMD8 genes connected with drug

metabolism RETSAT or genes with poorly

understood function FAM13C KANK3 and

CTSH


The cells in culture A549 (lung cancer)

have been treated with actinomycin D and

nutlin-3a In order to confirm the

hypothesis the gene regulatory region of

investigated genes with a potential p53

binding site has been cloned into pGL3-

Basic reporter vector Additionally we have

mutated the putative p53 binding site using

site-directed in vitro mutagenesis system

166

The results of RNA-seq have been validated

by Real-Time PCR The selected genes

validated by qRT-PCR will also be tested

for their dependence on p53 We will com-

pare their expression in control conditions

or following A+N treatment in A549 cells

with knocked-down p53 and in controls for

knockdown

Results

We have confirmed our hypothesis that p53

affects the induction of DRAXIN RETSAT

FAM13C KANK3 FRMD8 CTSH follo-

wing co-treatment with actinomycin D and

nutlin-3a The cloned promoters of invest-

tigated genes contain bona fide p53

response element


This recently identified new biological link

between p53 and immunity Wnt signalling

pathway drug metabolism and newly

discovered p53-regulated genes deserves

more detailed survey in further studies on

these new functions of p53 tumor sup-

pressor protein

References [1] Allen MA Andrysik Z et al Global analysis of

p53-regulated transcription identifies its direct

targets and unexpected regulatory mechanisms

Elife 2014 May 273e02200

[2] Tebaldi T Zaccara S et al Whole-genome

cartography of p53response elements ranked on

transactivation potential BMC Genomics 2015

Jun 1716464

[3] Zajkowicz A Gdowicz-Kłosok A et al

Actinomycin D and nutlin3a synergistically promote

phosphorylation of p53 on serine 46 in cancer cell

lines of different origin Cell Signal 2015

Sep27(9)1677-87

This work has been supported by grants

no 201727NNZ501079 to BŁ-S from the

National Science Centre Poland (NCN)


Anticancer activity of curcumin and wogonin in colon cancer cells

Magdalena Machynia1 Karolina Matuszewska

1 Helena Moreira

2 Ewa Barg

2

1Student Research Group of Flow Cytometry and Biomedical Research at the Department

of Basic Medical Sciences Faculty of Pharmacy Wroclaw Medical University 2Department

of Basic Medical Sciences Faculty of Pharmacy Wroclaw Medical University

Colon cancer affects a great number of

people each year and is a second leading

cause of cancer-related death in the world

Current oncotherapies are not fully effective

due to high degree of resistance of colon

cancer cells to cytostatic drugs There is

therefore a need for novel treatment options

that could overcome or avoid drug resis-

tance in this cancer Combination therapy

consisting of chemotherapeutic drugs and

natural polyphenol compounds could

improve the effectiveness of pharmaco-

logical treatment Curcumin and wogonin

are promising polyphenols for adjuvant

cure In this review we provide an overview

of their properties and mechanisms of action

on colon cancer with special emphasis on

theirs potential to improve the effectiveness

of cytotoxic drug

Curcumin a bright yellow natural dye is

the component of turmeric a common spice

which found use in many cuisines all around

the world Curcumin chemically known as

diferuloylmethane (C21H20O6) is derived

from rhizome of the Curcuma longa plant

from the Zingiberaceae family It was

demonstrated by many published researches

that curcumin possesses anti-toxic anti-

inflammatory antioxidant and potentially

chemotherapeutic properties The anticancer

effects of curcumin is associated by its

inhibition of proliferation and angiogenesis

in cancer cells and induction of apoptosis

167

Wogonin a monoflavonoid with chemical

formula of C16H12O5 is natural substance

located in the Scutellaria baicalensis radix

the plant from the Lamiaceae family The

activity of the wogonin supported by

numerous studies includes anticancer anti-

inflammatory antioxidant and neuropro-

tective properties In recent studies it was

also showed that wogonin can display

anxiolytic effects The antitumor action of

wogonin involves induction of apoptosis

and cell differentiation and inhibition of

several genes important for regulation of the

cell cycle

The chemopreventive activity of curcumin

and wogonin in several animal tumor model

systems strongly suggest their potential to

improve cytostatic therapy in colon

cancer It has been shown that curcumin

enhances the effects of irinotecan on colon

cancer cells Irinotecan is a key anticancer

drug used for the treatment of meta-

static colon cancer Chemosensitization

potential of wogonin in drug-resistant colon

cancer has also been demonstrated The

combining of an anticancer drug with

curcumin or wogonin may therefore

improve cytotoxic effects of monotherapy

and reduce undesirable side effects

References [1] BB Aggarwal A Kumar et al Anticancer Res

vol 23 (1A) 363-398 2003

[2] NG Vallianou A Evangelopoulos et al

Anticancer Res vol 35(2) 645-652 2015

[3] M Li-Weber Cancer Treatment Reviews vol

35(1) 57-68 2009 doiorg101016jctrv2008

09005

[4] MCh Tai SY Tsang et al CNS Drug Reviews

vol11(2)141-150 2005

doiorg10111 1j1527-34582005tb00266x

[5] YF Huang DJ Zhu et al Oncotarget

vol8(25)40264-40275 2017

DOI 1018632oncotarget16828

[6] H Moreira T Gębarowski et al Bromatol

Chem Toksykol vol48 (3) 467-472 2015


Analysis of dendrimer-protein interactions and their implications

on potential applications of dendrimers in nanomedicine

James Magnus Rae1 Barbara Jachimska

2

1Chemical and Process Engineering University of Strathclyde 2Jerzy Haber Institute

of Catalysis and Surface Chemistry Polish Academy of Sciences

Background

Dendrimers are three-dimensional nano-

sized polymers As these macromolecules

are synthesised there is a controlled increase

in the size molecular weight and number of

surface groups with the increase of each

increasing generation number The chemical

architecture of PAMAM dendrimers as well

as their small sizes makes dendrimers an

attractive proposition for research into their

medical applications[1]

PAMAM dendrimers are perfect candidates

as carriers for the delivery of anticancer

drugs due to them being highly soluble and

having many chemically versatile surface

groups These allow for conjugation of

anticancer drug molecules to develop

dendrimer based drug delivery [1 2]

The binding of protein to dendrimers can

alter the structure mobility conformation

and functional activity of the dendrimer

with electrostatic forces playing a predo-

minant role in the interactions between

dendrimers and protein In order to fully

evaluate the potential of dendrimers in

nanomedicine the impact of dendrimer-

protein interactions must be understood[3]


The study used G55 PAMAM The zeta

potential of the dendrimer was calculated by

168

the measurement of mobility from Electro-

phoresis the measurements were taken

at different values of pH this was repeating

using solutions prepared at differing ionic

strengths

The efficiency of G55 PAMAM adsorption

on gold surface and the properties of the

formed layer depending on the pH of

dendrimer solution was also determined

using a quartz microbalance with energy

dissipation monitoring (QSense E1 Biolin

Scientific) with a flow module The

adsorbed mass on the sensor for homo-

geneous rigid films was calculated using

the Sauerbrey model

The size of dendrimer monomers dependant

on varying pH was found in solution was

measured using dynamic light scattering at

differing ionic strengths

The absorbance and wavelength of the G55

PAMAM dendrimer was measured for

varying concentrations at constant ionic

strength using UV-Vis As well as for

constant concentration while varying pH for

solutions of different ionic strength

Results

From the results of this experiment it was

determined that the isoelectric point of G55

dendrimer was found to be at pH=5 It was

noted that the initial zeta potential at natural

pH (94-102) was negative and that as the

ionic strength of the solution prepared

increased the zeta potential at corresponding

pH values increased becoming less negative

The adsorption of G55 dendrimer onto

a gold surface under different conditions

(pH ionic strength and concentration) was

investigated using Quartz Crystal Micro-

balance (QCM-D) Results from this

experiment indicate that the maximum

adsorption occurred at pH=5 corresponding

to the isoelectric point of G55 dendrimer It

was found that on rinsing the gold surface

with solution of the same ionic strength with

a pH of 75 the dendrimer was seen to

desorb from the gold surface

The size of the of the monomers was found

to range from 66nm to 8nm with there

being an increase in size observed at

pH=75

The absorbance was found to be higher in

solutions with lower ionic strength with the

wavelength being higher in solutions with

higher ionic strength It was noted that the

that the absorbance and wavelength incre-

ased with increasing pH and with increasing

concentration


The G55 dendrimer was found to have

properties that would present an attractive

option as a drug carrier in nanomedicine

One barrier presented from the findings was

the desorption of dendrimer when rinsed

with solution of pH=75 The interactions of

G55 PAMAM with bovine serum albumen

and fibrinogen will be studied to determine

what effects the dendrimer-protein

interactions have on the secondary structure

of the protein

Acknowledgments

This work was partially supported by

project NCN OPUS 201623B02788

Erasmus+ UK GLASGOW 02

References [1] S H Medina and M E H El-Sayed

Dendrimers as carriers for delivery of chemo-

therapeutic agents Chem Rev vol 109 no 7 pp

3141ndash3157 2009 doi 101021cr900174j

[2] Q Ma et al Oral Absorption Enhancement of

Probucol by PEGylated G5 PAMAM Dendrimer

Modified Nanoliposomes Mol Pharm vol 12 no

3 pp 665-674 Mar 2015 doi 101021

mp500388m

[3] D Shcharbin et al Dendrimer-protein

interactions versus dendrimer-based nanomedicine

Colloids Surfaces B Biointerfaces vol 152 pp 414-

422 2017 doi 101016jcolsurfb201701041


169

YM-1 as a modulator of HSP70 protein in chemotherapy combined

with 5-fluorouracil

Kamila Majgier1 Julita Kulbacka

2

1Faculty of Pharmacy Wroclaw Medical University Wroclaw Poland 2Department of Molecular and Cellular Biology Wroclaw Medical University Wroclaw Poland

Background Recent studies have shown that molecular proteins such as heat shock protein 70 (Hsp70) can be a potential target of anti-cancer therapy Hsp70 is a stress inducible chaperon and can be accumulated in cells in effect of various stressing factors provo-king lethal conditions In cancer cells Hsp70 is involved in oncogenesis and resistance to chemotherapy Thus the inhibition of Hsp70 seems to be a promising anticancer approach [1] Here we proposed YM-1 molecule as an inhibitor of this protein In multiple cancer lines YM-1 was found to selectively target cancer cells over normal cells [2] There was proved that YM-1 can preferentially bind to the ADP-bound form of HSP70 and therefore an inhibitory effect was observed [3]

The aim of our study was to compare the effects of YM-1 interaction with 5-fluoro-uracil (5-FU) against human colorectal cancer cells (LoVo)

Material and methods Human adenocarcinoma cells (LoVo) were used as a model in vitro 5-fluorouracil was implemented as a standard chemothera-peutic in colon cancer YM-1 (2-[[3-Ethyl-5-(3-methyl-2(3H)-benzothiazolylidene)-4-oxo-2-thiazolidinylidene]methyl]-1-methyl-pyridinium chloride) was used as an inhi-bitor of Hsp70 Cell viability after exposi-tion to 5-FU YM-1 or combinations was evaluated by MTT assay after 24 and 48 hours Hsp70 expression was semi-quantita-tively determined by immunocytochemical method

Results We showed that YM-1 has anti-tumour activity in LoVo cells and that in combi-nation with 5-FU after longer incubation (48h) resulted in synergistic effect Im-munocytochemical studies revealed an alter-nated expression of Hsp70 in colon cancer cells in particular after exposition to YM-1 or YM-1 combined chemotherapy

Discussion The available data indicate a pivotal role of the HSP70 in regulation of apoptotic cell death in cancer cells Thus the modulation of chaperons appears an interesting approach in cancer treatment Some authors indicated a unique YM-1 mechanism of action cause its ability to destabilize Hsp70ndashBag3 acti-vities and finally to suppression of cancer-promoting signalling pathways [2] There was shown that another inhibitor ADD70 applied in animal models of colon cancer and melanoma showed promising effects on tumour size and growth and sensitized cancer cells to chemotherapy [4] Similarly we have also proved a synergistic effect of YM-1 molecule with 5-FU in colon cancer but further mechanism in this cancer type should be investigated

Funding The research was supported by National Science Centre (Poland) within a framework of SONATA BIS 6 (201622ENZ500671 PI J Kulbacka)

References [1] C Boudesco S Cause G Jego and C Garrido Methods in Molecular Biology vol 1709 Humana Press Inc 2018 pp 371-396

170

[2] T A Colvin et al Cancer Res vol 74 no 17 pp 4731-4740 Sep 2014 doi 1011580008-5472CAN-14-0747 [3] K M Zulfiker Rahman S Kose and N Imamoto 2017 doi 103191thermalmed33129

[4] E Schmitt et al Cancer Res vol 66 no 8 pp 4191ndash4197 Apr 2006 doi 1011580008-5472CAN-05-3778


Cytotoxic effect of Sculletariae baicalensis derived flavonoid baicalein

and ascorbyl palmitate in the treatment of Pancreatic Ductal

Adenocarcinoma

Markowski A1 Zaremba-Czogalla M

1 Gubernator J

1

1Faculty of Biotechnology Department of Lipid and Liposomes University of Wrocław

Background Pancreatic ductal adenocarcinoma (PDAC) is among the most deadliest and the worst to diagnose and treatment type of cancer It is predicted that by 2030 PDAC will be the second leading cause of cancer-assosciated deaths in the USA and it will surpass breast colon and prostate cancers in that matter [1][2] Currently available types of therapies based on gemcitabine and FOLFIRINOX are insufficient and it leads to a need for develop new ways of therapy and drug development for PDAC Lipo-somes are well known tools for enhancing bioavailabillity and pharmacokinetic of wide range of hydrophobic and hydro-phillic drugs [3] Due to enhanced perme-abillity and retention effect (EPR) which in PDAC occurs in high rate liposomes are potentially very good candidades in therapy of PDAC [4] Baicalein (BAI) is one of the Sculletariae baicalensis flavonoid com-ponent which have widespread of antiin-flammatory antiviral and anticancer pro-perieties [5] Ascorbyl palmitate (PalmAs) is lipid derivate of ascorbate which is known for its potential anticancer activities against PDAC [6] In this paper we exami-ned the cytotoxic effect of BAI and PalmAS towards two pancreatic cell lines with correlation between cytotoxicity on control cell line NHDF We have also combine these two drugs into single therapy Lastly we have prepared liposomal formulation of

PalmAs and BAI with high entrapment effciency for enhanced delivery

Material and Methods All phospholipids were purchased in Lipoid Germany Cholesterol was donated by Hasco-Lek Poland Baicalein was pur-chased in Haoxuan Bio China Ascorbyl palmitate was purchased in Gonmisol Spain All organic and non-organic solvents was purchased in Chempur Poland PalmAs and BAI liposomes was prepared using solvent-evaporation metod with compound lipid weight ratio 110 Obtained thin lipid layer was dissolved in tert-buthanol and freeze-dried overnight Obtained lipid cake was hydrated using 150 mM sodium chloride and calibrated via extrusion Size and polydispersity was measured using Malvern ZetaSizer NanoZS Concentration of PalmAS was measured via HPLC method Concentration of BAI was deter-mined by spectrophotometric methods Cytotoxicity was determined using MTT assay with BAI dissolved in DMSO and PalmAS as liposomal formulation Values of IC50 was calculated using GraphPad Prism 6

Results Size and polydispersity of PalmAs and BAI liposomes were suitable for intravenous application Entrapment efficiency was nearly 100 IC50 values for BAI in DMSO was 2983microM for BxPC-3 cells

171

and 2984microM for AsPC-1 cells For PalmAs in liposomes these values were 12473microM for BxPC-3 cells and 4084microM for AsPC-1 cells NHDF cell line was unaffected or affected in limited way only in the highest concentrations by those compounds We have also observed doubled cytotoxicity by using these compounds combination on both cancer cell lines with effect of this combination on NHDF cells for further investigation and determination

Discussion and conclusions Based on obtained data we believe that both of these compounds can be used in proposed liposomal therapy of PDAC Both com-pounds carry potent cytotoxic effect against PDAC cell lines with limited cytoxicity against normal cell lines Easy and efficient liposomal encapsulation process gives many prospects for future o potential of pharma-

ceutics suitable for intravenous admini-stration because of their enhanced pharma-cokinetics

References [1] Provenzano PP Cuevas C et al Cancer Cell 2012 Mar 2021(3)418-29 doi 101016jccr 201201007 [2] Lafaro KJ Melstrom LG Am J Pathol et al 2019 Jan189(1)44-57 doi 101016jajpath 201809009 [3] Natfji AA Ravishankar D et al J Pharm Sci 2017 Nov106(11)3179-3187 doi 101016jxphs201706019 Epub 2017 Jun 29 [4] Gubernator J Expert Opin Drug Deliv 2011 May8(5)565-80 doi 101517174252472011

566552 [5] Liu H1 Dong Y et al Int J Mol Sci 2016 17 1681 doi103390ijms17101681 [6] Polireddy K Dong R et al Sci Rep 2017 Dec 77(1)17188 doi 101038s41598-017-17568-8


Mechanisms of protective action of polyphenol extract Sea buckthorn

(Hippophae rhamnoides L) in relation to the erythrocyte membrane

K Męczarska1 S Cyboran-Mikołajczyk

1 N Gorczyca

1 O Antczak

1

S Lachowicz2 D Bonarska-Kujawa

1

1Department of Physics and Biophysics Wrocław University of Environmental and Life Sciences Wrocław Poland 2Department of Fermentation and Cereals Technology Wrocław University of Environmental and Life Sciences Wrocław Poland

Background Sea buckthorn extract is a rich source of nutrients antioxidants and other com-ponents that can potentially exert protective role in living organisms Attributed to it is an antioxidant and anti-inflammatory action helpful in treatment of tumors and the cardiovascular gastrointestinal and skin diseases [1-3] The studies were designed to determine the polyphenolic composition and biological activity of extract of Sea buckthorn (Hippophae rhamnoides L) in relation to erythrocyte membranes In the study the erythrocyte was treated as an example and model of the animal cell and its membrane as a simple model of bio-

logical membrane which is the first site of contact between physical agents and the body

Material and Methods Sea buckthorn extract was obtained from the Department of Fermentation and Cereals Technology Wroclaw University of Envi-ronmental and Life Sciences A detailed quantitative and qualitative analysis of extract was conducted using the chroma-tographic (UPLC-DAD UPLC-ESI-MS) and spectrophotometric (Folin-Ciocalteu) methods The biological activity of the extract was investigated in relation to erythrocytes and isolated membranes of erythrocytes by using spectrophotometric

172

and fluorimetric methods Spectrophoto-metric method was used to determine the effect of the extract on the degree of haemolysis and osmotic resistance of erythrocytes The antioxidant activity of Sea buckthorn extract towards erythrocyte mem-branes was determined with spectrophoto-metric and fluorimetric methods using two oxidation inducers UVC radiation and AAPH

Results A total of 40 polyphenolic compounds found Sea buckthorn preparation were identified The study confirmed high antioxidant activity of the polyphenols contained in Sea buckthorn extract

compared to that of Trolox The results of hemolytic investigations showed that Sea buckthorn extract does not induce hemo-lysis which means there is no destructive action on the erythrocyte membrane The osmotic resistance investigation of extract modified erythrocytes using hypotonic NaCl solutions did not show significant changes in the increase in osmotic resistance of erythrocytes

Discussion and conclusions Thanks to the rich polyphenolic compo-sition and high antioxidant activity Sea buckthorn extract protects the body against the harmful effects of free radicals High antioxidant activity classifies the tested extracts as a valuable source of compounds that can find wide application in the preven-tion and treatment of many diseases arising as a result of disturbed oxidative processes in the body

This study was supported by funds of the statutory activity of the Department of Physics and Biophysics of the Environ-mental and Life Sciences University of Wrocław

References [1] P Bośko WBiel Post Fitoter 18(1) 36-41 2017 [2] I Sytařovaacutea J Orsavovaacuteb et al Food Chemistry vol 310 2020 125784 DOI httpsdoiorg101016jfoodchem2019125784 [3] R Pariyani M Kortesniemi et al Journal of Food Science Vol 85 Iss 2 2020 DOI 1011111750-384115025


Unravelling the mechanisms of the increased efficacy of electrochemo-

therapy after catechin incubation in pancreatic cancer

Olga Michel1 Wojciech Szlasa

2 Mounir Tarek

3 Jolanta Saczko

1 Julita Kulbacka

1

1Department of Molecular and Cellular Biology Wroclaw Medical University Wroclaw Poland 2Faculty of Medicine Wroclaw Medical University Wroclaw Poland 3INC- SRSMC University of Lorraine ndash CNRS France

Background Catechins are green tea-derived polyphenols exerting the anticancer effects through a wide range of various mechanisms [1 2] In the previous study we have demonstrated that the co-treatment with catechin can firmly enhance the efficacy of electropo-ration (EP) with cisplatin in pancreatic cancer cells [3] We proposed that the effect can be attributed to non-transcriptional mechanisms evoked in cancer cells such as

the oxidative stress the impairment of multidrug resistance proteinsrsquo function and finally the direct impact on the membranersquos permeability Here we examined if the catechin influences cell permeabilization following the delivery of short electric pulses

Material and Methods The impact of catechin on electroper-meabilization was investigated using both theoretical and experimental approach The

173

molecular dynamics (MD) simulations were run and visualized with the GROMACS 20183 CHARMM-GUI and VMD software We have examined the localization of catechin and its influence on the thickness of the membrane Then we have compared the threshold electric field enabling perme-abilization with and without the catechinrsquos presence The model was verified experi-mentally by measuring the uptake of fluorescent dye YO-PRO-1 in pancreatic cancer cells in the presence of catechin using a flow cytometry method Finally we have performed the electrochemotherapy with calcium ions in two cancer cell lines (EPP85 181 RDB HPAFII) and control cell line (CHO-K1) incubated with catechin We have measured cell viability with the MTS assay

Results MD study revealed that during the exposure to the electric field catechin does not easily cross the lipid bilayer but rather localizes at the border of the water-lipid phase (Fig 1) affecting the membrane thickness Notwith-standing the latter catechin attachment had no effect on lowering the threshold electric field enabling pore formation This was confirmed experimentally as we did not observe increased uptake of YO-PRO-1 in the presence of catechin Finally unlike with the cytostatic compound [3] catechin

did not increase the efficacy of the EP with calcium ions in pancreatic cancer cells

Discussion and conclusions Although much attention has been given to anticancer properties of catechins in recent years data on their influence on bioelectro-chemical processes remain limited To our knowledge this is the first study to suggest that the increased efficacy of electroche-motherapy in vitro following the catechin incubation is not correlated with the increased permeabilization Another expla-nation of catechin action may be its interaction with the proteins responsible for drug resistance [4] Therefore next stage of the study is to investigate the relation between catechin incubation and the expres-sion and function of these proteins in pan-creatic cancer cells

This research project is financed by the National Science Center (Poland) No 201727NNZ301110 (to Olga Michel)

References [1] R Cooper DJ Morreacute et al Journal of Alternative amp Complementary Medicine vol 11 639-652 2005 DOI 101089acm200511639 [2] YYu Y Deng et al Apoptosis 19 1-18 2014 DOI 101007s10495-013-0908-5 [3] O Michel D Przystupski et al Acta Biochimica Polonica 65 173-1842018 DOI 1018388abp2018_2602 [4] D Przystupski O Michel et al Medicinal Chemistry Research 28 657-667 2019 DOI 101007s00044-019-02324-6

Fig 1 The formation of pore in the plasma membrane under the influence of the electric pulse

in the presence of catechin


174

1H nuclear magnetic resonance analysis of selected species

of methanogenic archaea

Karolina Anna Mielko1 Sławomir Jabłońśki

2 Piotr Młynarz

1 Marcin Łukaszewicz

2 1Department of Bioorganic Chemistry Faculty of Chemistry Wroclaw University of Science

and Technology 2Biotransformation Department Faculty of Biotechnology University

of Wroclaw

Background

NMR (nuclear magnetic resonance) has

become one of the primary methods used

for metabolomics studies in the last years In

addition to transcriptome and proteome ndash

omics studies metabolome analysis repre-

sents a third complementary approach for

identifying the metabolic pathways Meta-

bolomics studies involve an analysis of

microbial extracts (intra and extracellular)

In microbial metabolomics NMR is used

because it is nonselective quantitative and

reproducible method in metabolite quanti-

tative and qualitative analysis [1]

Methane fermentation involves a complex

series of metabolic reactions During this

processes organic compounds are broken

down into CO2 and organic acids which are

further degraded by acetogenic bacteria into

acetate CO2 and hydrogen All these sub-

strates are then preferentially utilized by

methanogenic bacteria to produce methane

Sometimes the strains possess the reduced

activity accompanied by decreased methane

gas production The reason of diminished

methanogenesis is not known so far

Therefore the changes in the intracellular

metabolites pathways of investigated

microorganisms should be monitored and

analysed [2]

The objective of this research is to present

the initial results obtained by the 1H NMR

analysis of methanogenic archaea metabo-

lome Individual goals of the preliminary

studies included type and efficiency of

extraction method growth conditions

influence relationships between different

species metabolites identification


Biomass samples were obtained from

NBRC (Japan) Lyophilized cells were

homogenized and extracted with methanol

water solvent system Then the samples

were evaporated and dissolved in PBS

buffer All samples were investigated by use

of 1H NMR spectroscopy together with

chemometric methods

Results

Based on obtained NMR spectra the PCA

model was calculated with two principal

components (PC) which revealed the

natural grouping of the various bacteria

strains Three distinct groups of samples

were formed Methanobacterium cluster M

acetivorans cluster and Methanosarcina

cluster (Fig 1)

In addition to untargeted PCA analysis also

supervised OPLS-DA analysis was perfor-

med according to internal parameters

medium substrates additional reaction

substrates and cultivation temperature

The assignments of metabolite were perfor-

med for all found intracellular metabolites


The obtained data by multivariate data

analysis of spectra revealed taxonomical

relationships between strains while change

in cultivation conditions has influence on

the metabolic profile of M acetivorans

Further analysis of metabolomic relations

between methanogenic archaea requires

a broader set of species (and repetitions for

175

each samples) More details regarding meta-

bolic pathways (including higher number of

identified compounds) would require wider

metabolome analysis Although data were

obtained on the limited number of samples

the results are auspicious Due to the slow

growth rates of individual archaea species

cultivation in chemostat conditions should

be considered to facilitate the biomass

collection

References [1] A K Kosmides K Kamisoglu et al Crit Rev

Biomed Eng vol41(3) 205-221 2013 DOI

101615critrevbiomedeng2013007736

[2] D Sasaki K Sasaki et al Bioresource

Technology vol 172 83-90 2014 DOI

101016jbiortech201408054

Fig 1 PCA score plot of methanogenic archaea


Differential effects of novel pyrimidine derivatives on hepatocarcinoma

cells viability

Aleksandra Mikołajczyk1 Aleksandra Wolska

1 Marcin Stolarczyk

2 Agnieszka

Matera-Witkiewicz1

1Screening Laboratory of Biological Activity Test and Collection of Biological Material

Faculty of Pharmacy Wroclaw Medical University 211A Borowska 50-556 Wroclaw

Poland 2Department of Organic Chemistry Faculty of Pharmacy Wroclaw Medical

University 211A Borowska 50-556 Wroclaw Poland

Background

Hepatocellular carcinoma (HCC) is the fifth

(men) and ninth (women) most commonly

occurring cancer in adult population HCC-

related mortality is still increasing in many

countries as the majority of patients is

present at an advanced stage of the disease

[1] New chemotherapeutic agents are

constantly synthesized many of which are

heterocyclic compounds Among many

biological activities they present the

anticancer potential is one of our main

interest

A novel series of pyrimidine derivatives

was synthesized In their structure they

contain an amino group at the position 4 and

a hydroxymethyl or carboxyl group

176

at the position 5 To the amino group

various substituents were attached

Our aim was to determine the antitumor

activity of novel pyrimidine derivatives on

human hepatoma cell line (HepaRG) by

defining their half maximal inhibitory

concentrations which inhibits biological

function (IC50)


For screening tests neutral red uptake assay

was performed according to the protocol

[2] Briefly compounds in appropriate

concentrations were introduced into 24-hour

cell culture After the specified period of

time cells were incubated with a neutral red

solution followed by fixation with a 50

ethanol solution containing 1 glacial

acetic acid The amount of uptaken neutral

red was measured spectrophotometrically at

540 nm

For flow cytometry experiments compounds

with lowest antitumor concentrations selec-

ted after screening tests were analyzed

After incubation cells were detached and

fluorescently stained with propidium iodide

(PI) and fluorescein diacetate (FDA) accor-

ding to the modified protocol [3]

Results

Five pyrimidine derivatives with different

substituents were tested Amongst them

three had a hydroxyl and two had a carboxyl

group at the position 5 Compounds with

hydroxymethyl group at the position 5 had

lower IC50 than those with carboxyl group

at the same place Among the substituents at

the position 4 tert-butylamine group was

found to be the most effective cytotoxic

agent (Tab1)

Tab 1 IC50 values obtained in screening tests

for five pyrimidine derivatives Substituents at the

position 4 (s) are listed Hydroxyl and carboxyl

group at the position 5 are marked as () and ()

respectively

IC50 [μM]

s amino group () 590

s aminopropyl group () 600

s tert-butylamine group () 80



It is worth pointing out that cytotoxicity of pyrimidine derivatives concerns only cancer and not normal cells (viability of L929 cell line exceeded 90 in all tested concen-tration ranges)

Flow cytometry experiments provided infor-mation about the type of cell death

Discussion and conclusions The pyrimidine ring is a widely used structure in medicinal chemistry It has been demonstrated that such structures show many biological activities ie antimicrobial antiviral or antioxidant Many used drugs with potential anticancer activity contain pyrimidine core [4] Our newly synthesized pyrimidine derivatives show differential anticancer activity against hepatoma cell line Based on IC50 values the most promi-sing compounds had tert-butylamine group at the position 4 with slightly better results for compound with hydroxyl group at the position 5 Further studies for precise mole-cular mechanisms of action and potential therapeutic usage should be undertaken

The research was founded by Wroclaw Medical University (project number SUBD25019012)

References [1] J D Yang et al Nature Reviews Gastro-enterology amp Hepatology vol 16(10) 589-604 2019 DOI 101038s41575-019-0186-y [2] G Repetto et al Nature Protocols vol 3(7) 1125-1131 2008 DOI 101038nprot200875 [3] K H Jones JA Senft J Histochem amp Cytochem 3377 1985 DOI 101177 3312578146 [4] N M Ahmed et al J Enzyme Inhib Med Chem vol 34(1) 1110-1120 2019 DOI 101080 1475636620191612889


177

Genotoxic effects of resveratrol celastrol and camptothecin in mono-

and combined therapy in colon cancer cells lines

Helena Moreira1 Małgorzata Grzesik

1 Benita Wiatrak

1 Anna Szyjka

1 Ewa Barg

1

1Department of Basic Medical Sciences Faculty of Pharmacy Wroclaw Medical University

Background Colon cancer is the most prevalent and lethal malignancies The major problem of successful treatment of this cancer is the existence of primary resistance andor the acquisition of secondary resistance to currently available cytotoxic drugs In vitro studies have found that some natural polyphenolic compounds including resve-ratrol and celastrol reduce chemo-resistance in different cancer cell lines Here we investigated the genotoxic activity of both polyphenols in combinatory therapy with camptothecin a key component of first- and second-line treatment regimens for meta-static colorectal cancer (CRC)

Material and Methods The study was carried out using doxorubicin resistant LOVODX) and drug sensitive colon cancer cell line (LOVO) The original LOVO cells were derived from a fragment of a metastatic tumor nodule of human colon adenocarcinoma (Dukesrsquotype c grade IV ATCCreg CCL-229trade) LOVODX cells were obtained by 3-month incubation of LOVO cells with low doses of doxorubicin The genotoxicity was assessed by the means of FHA method (Fast Halo Assay) The cells were incubated for 24 hours in the presence of tested drugs in mono- and combined-therapy The combination of 1 5 and 10 microM of Celastrol or 5 and 10 microM of resveratrol with 10 microM of camptothecin were used

Results In monotherapy an increase in the percentage of DNA damage was observed

in LOVO cells for both polyphenols by 20-60 depending on the concentration tested These effects were significantly stronger in LOVODX cells ie 2-25 fold increase in DNA double strand breaks (DBSs) was noted Combined treatment increased the genotoxic effect of celastrol when compared to camptothecin alone or celastrol alone These effects were additive In contrast combined resve-ratrol and camptothecin induced only a very slight increase in the percenteage of DBSs

Discussion and conclusions The results indicate that both celastrol and resweratrol demonstrate significant geno-toxic effect in colon cancer cells especially in cells expressing high level of drug resistance However celastrol appears to be a better candidate for adjuvant treatment in this type of cancer due to its synergistic effects with cytotoxic drugs

References [1] H Moreira A Szyjka et al Oxidative Med Cell Longev vol 2019 art6793957 2019 DOI 10115520196793957 [2] H Moreira A Szyjka et al Oncotarget vol 9 (30) 21211-21223 2018 DOI 1018632 oncotarget25014 [3] L Huang S Zhang et al RSC Adv vol 9 2572ndash2580 2019 DOI 101039c8ra08364a [4] M Honari R Shafabakhsh et al Cancer Cell Int vol 19 (180) 2019 DOI org101186s12935-019-0906-y

This work was supported by the Wroclaw Medical University Grant No SUB D13019040


178

High frequency effects on square wave electroporation efficiency

Arūnas Murauskas1 Gediminas Staigvila


2 Auksė Zinkevičienė

2

and Vitalij Novickij1


Vilnius Lithuania

Background

Electroporation is a phenomenon of

increased biological cell membrane perme-

ability facilitated by pulsed electric fields

(PEF) [1] From the perspective of pulse

generation electroporation induced electro-

transfer of molecules depends on the pulse

amplitude duration and the number of pul-

ses however recently alteration of the

pulsing frequency gained increased interest

and a new modality of electroporation

protocols was proposed It is based on high

frequency (gt05 MHz) unipolar sub-

microsecond pulse bursts which enable

manipulation of the electroporation effi-

ciency using pulse repetition frequency as

a sole parameter without change of the

energy of the burst [2]ndash[6] In this work we

have studied the capacitive charging of the

cell membrane to predict permeabilization

efficiency during high frequency electro-

poration


The finite element model of the biological

cell was developed in COMSOL environ-

ment (COMSOL Sweden) using an axisym-

metric geometry A free triangular mesh

with 27931 domain elements and 511 boun-

dary elements was formed [7]

During experiments Chinese Hamster

Ovary (CHO) cells CHO-K1 were used For

electroporation the 0ndash3 kV 60 A square

wave 100 nsndash1 ms pulse generator (VGTU

Vilnius Lithuania) with a commercially

available 1 mm gap electroporation cuvette

(Biorad Hercules USA) was applied

Bursts of 5 kVcm times 300 ns times 10 pulses

have been generated at three repetition

frequencies (1 Hz 10 kHz and 1 MHz) Cell

permeabilization efficiency was evaluated

using propidium iodide (PI Sigma-Aldrich)

and flow cytometry (Amnis Seattle USA)

Results

After low (1 Hz) and medium repetition

frequency (1 kHz) pulses electroporation

efficiencies were comparable (20plusmn5)

however 1 MHz protocol resulted in a dra-

matic increase of permeabilization efficiency

(70plusmn3) The number of permeabilized

cells increased solely due to the alteration of

the pulsing frequency without any changes

in the total energy of the burst which was in

agreement with the simulation model It was

shown that maintaining the transmembrane

potential (TMP) above the threshold during

the whole burst using MHz pulsing can

effectively improve permeabilization of the

cells By means of generating high-frequ-

ency pulse bursts it is possible to achieve

a threshold repetition frequency when the

discharging (TMP relaxation) time of the

membrane will be higher than the delay

time between the pulses thus the TMP

starts to accumulate throughout the burst

The simulation-predicted loss of the TMP

accumulation phenomena in the low and

medium frequency regions was also

occurrent experimentally

Conclusion

It was experimentally confirmed that the

new modality of unipolar high frequency

(MHz range) electroporation is a predo-

minantly polarization-based phenomenon

179

Acknowledgement


Council of Lithuania Grant Nr S-MIP-19-13




[2] V Novickij P Ruzgys A Grainys and S

Šatkauskas High frequency electroporation

efficiency is under control of membrane capacitive

charging and voltage potential relaxation

Bioelectrochemistry vol 119 pp 92-97 2018

[3] I Semenov M Casciola B L Ibey S Xiao

and A G Pakhomov Electropermeabilization of

cells by closely spaced paired nanosecond-range

pulses Bioelectrochemistry vol 121 pp 135-141

2018

[4] Z A Steelman G P Tolstykh H T Beier and

B L Ibey Cellular response to high pulse

repetition rate nanosecond pulses varies with

fluorescent marker identity Biochem Biophys Res

Commun vol 478 no 3 pp 1261-1267 2016

[5] A G Pakhomov et al Excitation and electro-

poration by MHz bursts of nanosecond stimuli

Biochem Biophys Res Commun 2019

[6] A Murauskas et al Predicting electrotransfer in

ultra-high frequency sub-microsecond square wave

electric fields Electromagn Biol Med pp 1-8

Dec 2019


Shear-wave elastography as a new diagnostic tool in evaluation

of masseter muscles stiffness

Anna Olchowy1

Mieszko Więckiewicz1 Andrzej Małysa

1 Piotr Seweryn

1 Joanna

Weżgowiec1 Joanna Smardz

1 Cyprian Olchowy

2

1Department of Experimental Dentistry Faculty of Dentistry Wroclaw Medical University

Poland 2Department of Oral Surgery Faculty of Dentistry Wroclaw Medical University

Poland aniaolchowygmailcom

Background

In modern high-end ultrasound machines

not only high resolution morphologic images

but also information on biomechanical pro-

perties of a tissue can be obtained The

ultrasound application named elastography

allows quantifying of the elasticity of the

musculoskeletal structures In particular

shear-wave elastography is considered to be

the most suitable type of ultrasound elasto-

graphy for the musculoskeletal system It is

widely used for tendons ligaments and

muscles [1-5]

Sonoelastography may be an optional

objective method used in diagnostics of

patients suffering from temporomandibular

disorders (TMD) which is an umbrella term

given to a variety of disease entities that

involves the masticatory muscles the

temporomandibular joints and associated

structures or both The etiology of temporo-

mandibular disorders is not completely

understood and it is considered to be

multifactorial according to the biopsy-

chosocial model Number of studies on

prevalence etiopathogenesis diagnostics

and management of temporomandibular

disorders have been published in recent

years

Aim of this study is a standardization of

sonoelastography measurement technique of

masseter muscles on healthy subjects without

TMD and determination of physiological

values of stiffness of masseter muscles


Thirty healthy volunteers with full dentition

or single tooth loss without previous history

of temporomandibular disorders and

without any deviations from physiological

function confirmed by the DC-TMD Axis I

protocol were included (19 females and 11

males) Patients were examined using shear-

180

wave elastography in prone position with

relaxed masticatory muscles In total the

stiffness of 60 masseter muscles were mea-

sured using Aixplorer Supersonics Mach 30

machine with 18 MHz linear probe Three

measurements of each masseter muscle

were performed Descriptive statistics were

used to analyse the collected data

Results

The mean stiffness of the masseter muscles

in healthy volunteers was 1072plusmn068 kPa in

males (the mean age 372plusmn29 years) and

1058plusmn125 kPa in females (the mean age

341plusmn22 years) Optimal measurement

technique of examination with probe

positioned parallel to the long axis of

muscle fibres and patient in prone position

was established


Shear-wave elastography proves to be useful

to quantify masseter muscles stiffness This

method provides reliable and reproducible

results Elasticity of masticatory muscles is

still a rather unexplored field of invest-

tigation with a potential to improve the

objective assessment of masticatory muscle

disorders

Acknowledgements

This work was financed by National

Science Centre ndash grant no PRELB160

18007 (A Olchowy)

References [1] Y Ariji A Katsumata et al J Oral Rehabil vol

36 627-635 2009 DOI 101111j1365-

2842200901977

[2] Y Ariji A Gotoh et al Oral Radiol vol 29

64-69 2013 DOI 101007s11282-012-0111-3

[3] Y Ariji M Nakayama et al Oral Surg Oral

Med Oral Pathol Oral Radiol vol 116 354-361

2013 DOI 101016joooo201305017

[4] Y Ariji M Nakayama et al Cranio vol 34

13-19 2016 DOI 1011792151090314Y

0000000037

[5] A Gotoh Y Ariji et al Oral Radiol vol 29

140-145 2013 DOI 101007s11282-012-0119-8


Klotho protein protects human cardiac myocytes from the damage

during ischemia and reperfusion

Agnieszka Olejnik1 Marta Banaszkiewicz

1 Anna Krzywonos-Zawadzka

1 Iwona

Bil-Lula1


Laboratory Hematology Faculty of Pharmacy with Division of Laboratory Diagnostics


Background

Heart ischemiareperfusion (IR) injury

causes an excessive formation of reactive

oxygen species (ROS) reactive nitrogen

species (RNS) degradation of contractile

proteins by proteolytic enzymes and necrotic

cell death of myocytes [1] Klotho is a

membrane-bound or soluble protein related

to aging Recent studies have proven pro-

duction of Klotho in cardiomyocytes and its

increased expression in stress-related heart

injury [2]

The aim of this study was to examine an

effect of Klotho protein on cell damage and

degradation of contractile proteins in the

cardiomyocytes subjected to IR injury

Material and Methods Human cardiac myocytes (HCM) was maintained in aerobic conditions in the control group In the study groups HCM were subjected to in vitro chemical IR (with sodium cyanide) in the presence or absence of recombinant human Klotho protein Lactate dehydrogenase (LDH)

181

activity served as a marker of cell injury The level of oxidative and nitrative stress and the degradation of contractile protein myosin light chain 1 (MLC1) were assessed

Results LDH activity was significantly higher (plt005) in cells subjected to IR compared to aerobic group Incubation of HCM with Klotho protein significantly decreased (plt005) cell injury during IR Total ROS and RNS activity was statistically higher (plt005) in IR group in comparison to aerobically maintained cells Klotho protein reduced the production of ROSRNS (plt005) and enhanced total antioxidant capacity (plt005) in cells subjected to IR An expression of inducible nitric oxide synthase (iNOS) gene was significantly lower (plt005) and the level of nitrate nitrite (NOx)

ndash was significantly higher (plt005) in myocytes from IR group in comparison to aerobic group The expres-sion of iNOS gene negatively correlated with LDH level (plt005 r= -078) ROSRNS activity (plt005 r= -049) and (NOx)

ndash level (plt005 r= -051) suggesting limited iNOS gene expression due to overproduction of nitratenitrite during injury Administration of Klotho protein reduced (NOx)

ndash level (plt005) and increased expression of iNOS gene (plt005) in cells subjected to IR The amount of MLC1 in

cell supernatants positively correlated with ROSRNS activity (plt005 r= 036) which indicates degradation and release of MLC1 during IR injury The degradation of MLC1 was significantly lower (plt005) in HCM subjected to IR in the presence of Klotho

Discussion and conclusions Reduction of oxidative and nitrative stress in cardiomyocytes injured by IR suggests potential cardioprotective effect of Klotho protein Klotho decreased cell damage and degradation of contractile proteins caused by IR thus Klotho may serve as a potential preventiveprotective agent during ischemic injury of the heart

References [1] I Bil-Lula H Lin et al Subthreshold nitric oxide synthase inhibition improves synergistic

effects of subthreshold MMP‐2MLCK‐mediated cardiomyocyte protection from hypoxic injury J Cell Mol Med 2016 20(6) 1086-94 DOI 101111jcmm12827 [2] S Song LY Si Klotho ameliorated isoproterenol-induced pathological changes in cardiomyocytes via the regulation of oxidative stress Life Sci 2015 135 118-123 DOI 101016jlfs201505024


Science Centre grant number UMO‐2016 23BNZ303151


Fruits of interspecific hybrids grapevines as a source of polyphenolic

compounds

Remigiusz Olędzki12

Nathan Tancula1 Karol Banaś

1 Agnieszka Orkusz

12 Joanna

Harasym12

1Faculty of Production Engineering Wrocław University of Economics and Business Poland 2Adaptive Food Systems Accelerator ndash Science Centre Wrocław University of Economics and Business Poland

Background Modern consumers are paying more and more attention to the presence of bioactive substances in food that can have a beneficial

effect on the human body Such substances include polyphenolic compounds which are attributed to among others anti-inflam-matory anti-virus or anti-cancer properties

182

[1] It has been confirmed that eating foods rich in polyphenolic compounds helps reduce the risk of developing civilization diseases (eg atherosclerosis diabetes cata-racts or Alzheimers disease) [2] There is a growing interest in polyphenolic com-pounds present in various varieties of hybrid grapevines (interspecific hybrids) which are grown in the temperate climate zone of the northern hemisphere

The aim of the study was to assess the total

content of phenolic compounds and the

content of flavonoids in the fruits of selec-

ted hybrid grapevine varieties (interspecific

hybrids) grown in ecological conditions in

Poland


The research used fruit of 5 grapevines

varieties Michigan Minesota V 68021

Beta and Alwood which were grown in the

vicinity of Jarosław in Poland The total

content of polyphenolic compounds fractions

(skin juice and pulp) was determined by

colorimetric assay using the Folin-Ciocalteu

reagent [3] The total flavonoids content

of the grapevine fruits was determined

spectrophotometrically using aluminum

chloride [3]

Results

Significant differences were observed in the

content of polyphenolic compounds (inclu-

ding flavonoids) in individual parts of the

grapevine fruit as well as in particular

grapevine varieties The highest content

of polyphenolic compounds in juice was

recorded in the fruit of the Michigan variety

(146 mg GAml) while the lowest - in the

fruit of the Alwood variety (017 mg

GAml) For example in the grapevine skin

of the Alwood variety the flavonoid content

was 264 mg quercetin100 g dry matter

whereas for the Michigen variety it was 92

mg quercetin100 g dry matter


The obtained results suggest that the

examined hybrid grapevine interspecific

hybrids grown in Poland may be a rich

source of phenolic compounds (including

flavonoids) The research suggests that due

to the content of bioactive compounds the

tested hybrid grapevine fruits can be

considered as an important component of

human diet with a potentially highly

beneficial effect on human health

References

List your references here and use the

example below

[1] Xia E Deng G Int J Mol Sci vol 11(2)

622-646 2010

[2] Balea ŞS Pacircrvu AE et al Oxid Med Cell

Longev vol 81 94-97 2018 doi

10115520188194721

[3] Vogiatzi G Tousoulis D et al Hellenic

J Cardiol vol 50 402-409 2009

[4] Fukumoto LR Mazza G J Agric Food

Chem vol 48 3597-3604 2000


Antioxidant activity and reducing power of hybrid grapevines fruit

grown in Poland


Karol Banaś1 Nathan Tancula

1 Agnieszka Orkusz

12 Joanna

Harasym12




183

Background

The special bioactive properties as well as

the taste make the grapevine fruit (Vitis L)

widely used in the food industry Due to the

fact that there is a lot of highly processed

food products on the food market the

interest in unprocessed natural food is

growing among consumers in Poland

Changing dietary habits of consumers

(looking for low-processed products with

high health-promoting quality) cause that

the fruit of hybrid grapevine varieties play

an increasingly important role in everyday

nutrition [1]

The aim of the study was to assess the

antioxidant and reducing activity and the

total content of fruit of selected hybrid

grapevine varieties (interspecific hybrids)

grown in ecological conditions in Poland

The study examined the fruit of 5 grapevine


Beta and Alwood which were cultivated in

the vicinity of Jarosław in Poland


The antioxidant activity of the grapevine

fruit fraction (skin juice and pulp) was

determined spectrophotometrically using

22-azinobis-3-ethylbenzothiazolin-6-sulfonic

cation radical (ABTS) and using 22-

diphenyl-1-picrylhydrazyl radical (DPPH))

The reducing properties of the grapevine

fruit were also assessed based on the FRAP

test [2-4]

Results

Significant differences in antioxidant

activity were observed in individual parts of

the grapevine fruit as well as among

varieties The antioxidant activity of fresh

grapevine fruit juice was of 0069 μM Trx

ml-1 for Alwood variety and 0754 μM Trx

ml-1 in Beta variety Statistically signi-

ficantly higher antioxidant activity was

observed in the grapevine varieties Beta V

68021 and Michigan compared to the

Minesota and Alwood varieties Obtained

research results suggest that in terms of

health the most valuable properties may

have the Michigen variety V 68021 and

Beta


The obtained research results suggest that in

terms of bioactive and health-promoting

properties selected grapevine varieties

(Michigen V 68021 and Beta) can be con-

sidered as highly valuable products with

potentially beneficial effects on the human

body

References [1] Minussi RC Rossi M et al Food Chem vol

82 409-416 2003

[2] Benzie IF Strain JJ Anal Biochem vol 239

70-76 1996

[3] Re R Pellegrini N Proteggente A et al Free

Radic Biol Med vol 26 1231-1237 1999

[4] Hengst C Werner S et al Eur Food Res

Technol vol 230 249‐254 2009


Analysis of relationship between polymorphism rs11640851 in MT1A

gene and MT Cu and Zn concentration in the AP patients group

Ołdakowska Monika Ściskalska Milena Milnerowicz Halina

Faculty of Pharmacy Department of Biomedical and Environmental Analyses Wroclaw

Medical University

Background

The acute pancreatitis (AP) is common

illness among gastrointestinal diseases in

many countries [1] The pathogenesis of AP

still has not been fully understood It is be-

lieved that one of the major pathogenesis of

AP is oxidative stress [2]

184

Metallothioneins (MTs) are cysteine rich

low molecular mass (6ndash7 kDa) proteins

They plays a role as the antioxidant defen-

ses MTs have thiol groups of cysteine

residues which have the highest affinity to

zinc (Zn) copper (Cu) [3] They take part in

metalloregulatory processes and control

cellular homeostasis of zinccopper MTs

are important for proliferation differen-

tiation and protection cells and tissues from

free radicals [4]

In single nucleotide polymorphism (SNP)

rs11640851 in MT1 gene is change from

threonine to asparagine (from C to A

nucleotide) [5] This change is located in

coding region in β domain It could have an

important influence on CuZn balance and

MT level in organism [5]

Aim of study

The aim of the project is to assess the

impact of genetic polymorphism

rs11640851 in MT1A gene on MT Cu and

Zn concentration in the blood of AP

patients


Polymorphism in MT1A gene was studied

by polymerase chain reaction (PCR) and

restriction fragment length polymorphism

analysis (PCR-RFLP) The results of the

PCR product were visualized by electro-

phoresis in agarose gel

Measurements of metals (Cu and Zn)

concentrations in serum will be determined

by Flame Atomic Absorption Spectrometry

method (FAAS)

The concentration of MT was measured

in erythrocyte lysate and plasma using two-

step direct ELISA method elaborated in our

laboratory [6]

Results

It was observed decrease Zn concentration

in the blood of non-smokers with CA

genotype compare to non-smokers with AA

and CC genotype It was shown a decrease

Zn concentration in the blood of smokers

with CA and AA genotypes compare to

smokers with CC genotype

It was shown that CuZn ratio was incre-

ased in the group of smoking AP patients

compare to non-smoking AP patients for

CA AA and CC genotypes

Discussion

This study was shown that SNP

rs11640851 in MT1A gene could play

important role in zinc homeostasis It was

confirmed that this genetic variation was

associated with reduced intracellular zinc

ion availability Polymorphism in MT1A

gene can contribute to decrease Zn concen-

tration leading to imbalance in CuZn ratio

Conclusion

A decrease in zinc concentration in the

blood of AP patients is associated with

polymorphism rs11640851

Abbreviations

AP acute pancreatitis MT metallothionein

SNP single nucleotide polymorphism Cu

copper Zn zinc

References [1] D Yadav A B Lowenfels Gastroenterology

144(6) 1252-1261 2013 doi101053jgastro

201301068

[2] SG Bareto Pancreatology 16(2) 157-63

2016 doi 101016jpan201509002

[3] H Milnerowicz M Jablonowska et al

Pancreas 38(6) 681-8 2009 doi 101097

MPA0b013e3181a53d1

[4] B Ruttkay-Nedecky L Nejdl et al Int J Mol

Sci 14(3) 6044-6066 2013 doi103390ijms

14036044103390ijms14036044

[5] M Raudenska J Gumulecet al Metallomics

6(1) 55-68 2014 doi 101039c3mt00132f

[6] H Milnerowicz A Bizoń Acta Biochimica

Polonica 57(1) 99-104 2010 doi 1018388

abp2010_2379


185

Evaluation of the diet of preschoolers from the Lower Silesian

Province

Orkusz Agnieszka12

Tancula Nathan2 Banaś Karol

2 Olędzki Remigiusz

12

Harasym Joanna12

Szydełko Monika2

1Adaptive Food Systems Accelerator ndash Science Centre Wroclaw University of Economics

and Business Poland 2Production Engineering Department of Biotechnology and Food

Analysis Wroclaw University of Economics and Business Poland

Background

Proper nutrition of children guarantees their

proper mental physical and social deve-

lopment It consists in providing the body

with all nutrients in appropriate quantities

and proportions From an early age children

should acquire healthy eating habits and

meals prepared in the kindergarten should

be balanced and follow standards Kinder-

garteners aged 4-6 years are particularly

vulnerable to dietary mistakes which can

cause a lot of diseases later in life Improper

supply of energy and nutrients can contri-

bute to disorders in the physical and mental

development of the childs body For this

reason it is very important to plan meals

based on nutritional standards The time that

children spend in kindergarten is 6-7 hours

per day During this period they usually eat

3 meals which should cover 75 of the

whole days energy and nutrient require-

ments The aim of the study was to assess

the energy and nutritional value of meals

served in the kindergarten based on

theoretical analysis of the menus using the

computer programme Dietetician 2


The assessment was made by using the

quantitative and qualitative methods com-

paring the value of energy and selected

nutrients (proteins fats carbohydrates

vitamins A B1 B2 C E mineral ingre-

dients Ca Fe) in the analyzed menus (ten

for each season of the year spring summer

autumn and winter) with the demanded

standards for children aged 4-6 years [1]

Results

The qualitative evaluation of the menus

showed that the breaks between meals were

not appropriate the meals were prepared

using various techniques salt consumption

was reduced by replacing it with herbs

seasonal vegetables and fruit were used in

the dishes In spring summer and winter

the energy value of the food ration was

adequate and within the limits of the

standards while in autumn it was above the

standards The quantitative analysis showed

an adequate protein intake in relation to the

recommended standards Fat content only

in autumn was within the range of accepted

standards in the remaining seasons of the

year it was too low Carbohydrate content

was too high in relation to the recommended

standards in all seasons of the year except

for winter The examined menus did not

cover the demand for selected mineral

components The content of calcium and

iron in each season was too low in relation

to the recommendations The results of the

analysis of menus showed that vitamins A

B2 and C in all seasons of the year

exceeded the accepted standards The

supply of vitamin E in the analyzed menus

was at a correct level in all seasons of the

year The analyzed menus covered the

demand for vitamin B1 only in summer In

the remaining seasons of the year the supply

of this ingredient was too high in relation

to the standards

186


The supply of carbohydrates exceeded

norms in all seasons of the year except

winter Too high carbohydrate level in

childrens diets can contribute to overweight

and obesity The content of vitamin B1

ranged from 048 mg in summer to 073 mg

in spring and only in the summer the supply

of this vitamin was within the range of

accepted standards and in the remainings it

was too high Since thiamine has a limited

ability to be absorbed from the gastroin-

testinal tract into the human body there is

no risk of adverse effects of its excessive

consumption Excessive consumption was

found for vitamins C B2 and A In case of

excessive intake of ascorbic acid there is no

clear evidence of adverse effects however

attention should be paid to the risk of

gastrointestinal disorders The high amount

of vitamin B2 in the studied menus was the

result of frequent offering to children a meat

and meat products (ham and sausages) In

the analyzed food rations the content of

vitamin A exceeded the norms almost twice

Excess vitamin A in the body may be

manifested by headache increased excita-

bility vomiting and skin changes Consu-

ming this vitamin in large quantities leads

to a loss of calcium from the bones and thus

to a decrease in bone mineral density which

results in osteoporosis The calcium and

iron content below the norms did not differ

significantly depending on the season of the

year The low amount of calcium in the diet

is unfavourable especially in children in

whom chronic calcium deficiency may

cause rickets With prolonged calcium

deficiency in the diet there is a decrease in

peak bone mass which in turn can result

in osteopenia To increase the amount of

calcium in the preschoolers diet more milk

and dairy products (cheese cottage cheese

and natural yogurt) should be given

The results of the study indicate the need for

intensive education about the proper meal

composition among employees of educa-

tional institutions (kindergartens)

References [1] Jarosz M (red) Normy żywienia dla populacji

Polski Warszawa 2017 httpsncezplupload

normy-net-1pdf


Box Diet ndash Can You Rely on It

Orkusz Agnieszka12

Banaś Karol2 Tancula Nathan


12

Harasym Joanna12

Dobrzyńska Daniela2




Background

In recent years the pace of human life has

increased significantly Usually over-

worked people do not have time to prepare

five meals a day by themselves and use the

services of catering companies offering

ready-made meals that are delivered directly

to the clients place of work or residence in

the form of a box Consumers ordering diets

through dietary catering companies are

convinced that they are in the hands of

professionals Usually on the websites of

companies offering boxed diets you can

find information that the diets are tasty and

healthy The word healthy should guaran-

tee that the diets are arranged in accordance

with the rules of menu arrangement and

balanced appropriately

187

The aim of the study was to evaluate the

diet of individual customers on the basis of

the standard diet developed by one of the

companies (the company was randomly

chosen) engaged in dietary catering based

in the Lower Silesia Province


This evaluation was carried out on the basis

of menus prepared for ten days For this

purpose the Diet 6D programme developed

by the Institute of Food and Nutrition in

Warsaw was used The energy value and

content of selected nutrients in each of the

ten menus was calculated

Results

The analysis of the diet catering menus

showed that they were not composed

correctly The shares of basic nutrients in

the standard dietary menus adopted by the

catering company did not comply with the

recommendations for a rational diet Too

low a percentage of carbohydrates and too

high a percentage of fats were found The

analyzed menus also contained too low

amounts of potassium and iron in

comparison with the standards The content

of protein salt sodium vitamins A C and

B2 exceeded the standards The amount of

dietary fiber magnesium and thiamine in

the diet was in accordance with the

standards The advantage of the served

meals was the fact that every day customers

were offered a variety of meals rich

in vegetables and fruit


It should be emphasized that on its website

the company described its diets as healthy

while at the same time offered rations that

were not properly balanced and arranged in

accordance with the rules of food prepa-

ration It is therefore necessary to make

consumers aware that the diet catering

service does not always meet their expec-

tations


Efficacy of biofilm eradication of Staphylococcus aureus strains

isolated from wounds by the antimicrobials commonly applied to treat

wound infections

Justyna Paleczny1 Malwina Brożyna

1 Karolina Dydak

1 Adam Junka

1 Marzenna

Bartoszewicz1


University

Background

The Staphylococcus aureus highly cross-

linked biofilm matrix is a barrier for

antibacterial compounds contributing to

persistent prevalence of this bacterium

in wound infections [1] The antimicrobial

agent for treatment of wound biofilm should

be selected with regard to the microbial

species being etiological factor of infection

its ability to form biofilm resistance to

antiseptics antibiotics and possible side

effects [2 3] The aim of this study was to

compare the efficacy of antibacterial agents

commonly used to treat wound infections in

eradication of Staphylococcus aureus biofilm

isolated from wounds


11 strains of Staphylococcus aureus

(including reference ATCC 6538 and

ATCC 33591 and 9 clinical strains) were

used for experimental purposes The

gentamicin antibiogram was performed in

accordance with EUCAST guideline from

2017 The antibiofilm activity of the

188

antibacterial agents was examined using

Minimum Biofilm Eradication Concen-

tration (MBEC) method The following

antimicrobial agents were scrutinized

polyhexamethylene biguanide with betaine

PHMB (01) povidone iodine PVP-I

(75) sodium hypochloritehypochlorous

acid solution NaClOHClO (001) and

gentamicin GENT (01) The drop of

biofilmsrsquo metabolic activity followed by

exposure on antimicrobial agents was

measured using the Richards method

Statistical analysis of results obtained was

performed by Statistica version 13 using

Shapiro-Wilk test Levenersquos test and U-test

with p-value 005 All tests were performed

in 2 repetitions and 3 replications

Results

While antibiogram results revealed that all

tested strains were susceptible to genta-

micin application of 01 of this antibiotic

did not lead to complete biofilm eradication

in case of 1011 strains Also 611 biofilm-

forming strains proved to be resistant to

001 HOCLNaOCl solution Therefore

these compounds were not taken into

account in statistical analysis The average

dilution of PHMB-based and PVP-I-based

products which enabled the complete

biofilm eradication was 35 and 22

respectively It translated into 036 gL of

PHMB and 1705 gL of PVP-I active

substances Both differences were statis-

tically significant


The tested agents used in the treatment of

wound infections have shown various

efficacy against staphylococcal biofilm

Gentamycin and hypochlorite compounds

showed the lowest effectiveness While

strains were sensitive to gentamicin (accor-

ding to antibiogram-based methodology)

the biofilm formed by the same strains

displayed high resistance against genta-

mycin antibiotic Our results stay in line

with data provided by another research team

[4]

Twice-diluted NaOClHOCl agent showed

no antibiofilm activity however other rese-

archers shown that higher concentrations of

hypochlorites may display such feature

antimicrobial Nevertheless such concen-

trations are also harmful to fibroblasts cells

responsible for wound healing [5]

Substances containing polyhexamethylene

biguanide with betaine and povidone iodine

were the most effective Even low concen-

trations of agents allowed to eliminate

staphylococcal biofilm However the

application of iodine povidone has certain

limitations It cannot be administered to

patients with hypersensitivity to iodine

hyperthyroidism or Duhring syndrome The

broad antimicrobial spectrum of PHMB and

rare side effects indicate on possible use

of PHMB-containing agents as a first-choice

agent in the treatment of wound infections

[2 3]


Wroclaw Medical University statutory

research SUBD23020002 and STM

D23020127

References [1] D E Moormeier K W Bayles Molecular

Microbiology vol 104(3) 365-376 2017

doi101111mmi13634

[2] M Bartoszewicz T Banasiewicz et al Forum

Zakażeń vol 10(1) 1-30 2019 dxdoiorg

1015374FZ2019002

[3] A Kramer J Dissemond et al Skin

Pharmacology and Physiology vol 31 28-58

2018 httpsdoiorg101159000481545

[4] B Maczynska A Secewicz et al PloS One vol

14(6) 2019 httpsdoiorg101371journal

pone0217769

[5] A L Severing J D Rembe et al Journal of

Antimicrobial Chemotherapy vol 74(2) 365-372

2019 httpsdoiorg101093jacdky432


189

Isobavachalcone increases doxorubicin accumulation in resistant colorectal cancer cells HT29Dx

Anna Palko-Łabuz Kamila Środa-Pomianek Maria Błaszczyk Anna Uryga Krystyna Michalak Olga Wesołowska

1

Department of Biophysics and Neuroscience Faculty of Medicine Wroclaw Medical University

Background According to the World Health Organi-zation cancer is one of the worldrsquos largest health problems It is estimated that almost 10 million people died prematurely as a result of cancer in 2017 One of the major obstacle in the successful therapy of neoplastic diseases is multidrug resistance (MDR) Such phenomenon may be the result of several mechanisms involving various aspects of cell biology However a pivotal role in the insensitivity of cancer cells to drug is played by ABC pumps These proteins are located in the membranes and decrease drug concentration within the cell Among ABC transporters P-glyco-protein (P-gp) is the best known mediator of MDR phenotype and its inhibition could improve cancer treatment Natural products are very important source of promising leads for the development of novel chemo-sensitizers [1] In our work we focused on the activity of isobavachalcone ndash phyto-chemical derived from Psoralea corylifolia Previous studies shown that antiproliferative effects of isobavachalcone against human cancer cells may be related to its inhibition of Akt signaling [2]

Material and Methods Cell based assays were performed on colorectal cancer cells HT29 and MDCK cells (Madin-Darby canine kidney) and their sublines characterized by P-gp overex-pression (HT29Dx and MDCK-MDR1) Cytotoxic activity was studied using sulpho-rhodamine (SRB) method In order to mea-sure doxorubicin accumulation fluorescence microscopy was applied The interaction of isobavachalcone with lipid bilayer was

investigated by differential scanning microcalorimetry (DSC)

Results We did not observe antiproliferative activity of isobavachalcone in HT29 and HT29Dx However using MDCK model cells we found that this compound was a substrate for P-gp These results were also confirmed in the experiments with P-gp inhibitor The application of doxorubicin ndash drug that is effectively removed from the cell by P-gp pump ndash together with isobavachalcone allowed for the increased uptake of the cytostatic by HT29Dx cells Also our studies demonstrated that isobavachalcone changed biophysical properties of lipid bilayer

Discussion and conclusions The results indicated that isobavachalcone was the substrate of P-gp and had ability to increase doxorubicin accumulation in drug-resistant cells Thus the chalcone might be a candidate for MDR-reversing agent Our work suggested that MDR reversing potential of isobavachalcone could be the result not only of its interaction with P-gp but also the ability to modify the lipid bilayer ndash the natural environment of ABC transporters

References List your references here and use the example below

[1] G Lee JY Joung et al Evid Based Complement Alternat Med 3412074 2018 DOI number 10115520183412074 [2] H Jing X Zhou et al Cancer Lett 294(2) 2010 DOI number 101016jcanlet201001035


190

What is the influence of ketogenic diet on the state of gut microbiota

Palma J1 Komorniak N

1 Kikut J

1 Kałduńska J

1 Żwierełło W

2 Skoacuterka-

Majewicz M2 Styburski D

2 Kapczuk P

3 Konecka N

4


in Szczecin Broniewskiego 24 Szczecin 71-460 Poland 2Department of Medical

Chemistry Pomeranian Medical University in Szczecin Powst Wlkp 72 Szczecin 70-111

Poland 3Department of Biochemistry and Medical Chemistry Pomeranian Medical

University in Szczecin Powst Wlkp 72 Szczecin 70-111 Poland 4Depertament

of Neurocognitive Science Pomeranian Medical University in Szczecin

Abstract

The ketogenic diet (KD) whose main

principles are high supply of fat and low

supply of carbohydrates has become

increasingly popular in recent years [1 2]

Initially KD was used to support the

therapy of illnesses with a neurological

basis but nowadays it is also increasingly

often used as a restriction and alternative

diet in sportsmen [1-6 8] Because KD can

lead to undesirable consequences the

International Ketogenic Diet Study Group

issued recommendations indicating groups

of patients that can follow this nutritional

plan These recommendations comply with

the directives of the National Centre for

Nutritional Education [3 9]

The most recent studies (in the last 5 years

PubMed Scopus) [6 7 10] reveal that KD

influences the composition of gut micro-

biota KD reduces the diversity of gut

microflora while causing an increase in

positive bacteria instead of pro-inflam-

matory bacteria [6 7] Studies that were

initially conducted on animals have now

been confirmed in humans [10] The studies

revealed a significant decrease in the

colonisation of intestines by Bacteroidetes

spp and an increase in Firmicutes spp and

Proteobacteria spp

Microbiome diversity was observed eg in

infants with drug-resistant epilepsy where

KD was applied We now know that gut

microbiome plays a crucial role in main-

taining the integrity of gut barrier It

modulates energy metabolism and prevents

inflammations observed in numerous

illnesses [11 12 16]

References [1] Stachowska E Palma J et al Wybrane diety

alternatywne i koncepcje żywieniowe In Dietetyka

sportowa Red Barbara Frączek Jarosław Krzy-

wański Hubert Krysztofia PZWL Wydawnictwo

Lekarskie Warszawa 2019 vol 1 p 723-760

[2] Veech RL The therapeutic implications of

ketone bodies the effects of ketone bodies in

pathological conditions ketosis ketogenic diet

redox states insulin resistance and mitochondrial

metabolism Prostaglandins Leukot Essent Fatty

Acids 200470(3)309-19

[3] Kossoff EH Zupec-Kania BA et al Optimal

clinical management of children receiving dietary

therapies for epilepsy Updated recommendations

of the International Ketogenic Diet Study Group

Epilepsia Open 20183(2)175-192 doi

101002epi412225

[4] Leone A De Amicis R et al Food and Food

Products on the Italian Market for Ketogenic

Dietary Treatment of Neurological Diseases

Nutrients 201911(5) doi 103390nu11051104

[5] Olson CA Vuong HE Yano JM Liang QY

Nusbaum DJ Hsiao EY The Gut Microbiota

Mediates the Anti-Seizure Effects of the Ketogenic

Diet Cell 14 czerwiec 2018173(7)1728-1741e13

[6] Zhang Y Zhou S et al Altered gut microbiome

composition in children with refractory epilepsy

after ketogenic diet Epilepsy Res 2018145163-

168

[7] Ma D Wang AC et al Ketogenic diet enhances

neurovascular function with altered gut microbiome

in young healthy mice Sci Rep 20188(1)6670

doi 101038s41598-018-25190-5

191

[8] Kraeuter AK van den Buuse M et al

Ketogenic diet prevents impaired prepulse

inhibition of startle in an acute NMDA receptor

hypofunction model of schizophrenia Schizophr

Res 2018 pii S0920-9964(18)30662-5

[9] Pol K Dieta ketogenna może być stosowana

wyłącznie w terapii oraz pod ścisłą kontrolą

dietetyka i lekarza httpsncezpl (Update

01122019)

[10] Spinelli E Blackford R Gut Microbiota the

Ketogenic Diet and Epilepsy Pediatr Neurol Briefs

20183210

[11] Adolph TE Grander C et al Liver-

Microbiome Axis in Health and Disease Trends

Immunol 201839(9)712-723 doi 101016

jit201805002 Epub 2018 May 26

[12] Tilg H Cani PD et al Gut microbiome and

liver diseases Gut 201665(12)2035-2044

[16] Han R Ma J et al Mechanistic and

therapeutic advances in non-alcoholic fatty liver

disease by targeting the gut microbiota Front Med

2018 doi 101007s11684-018-0645-9


Effect of acrylamide supplementation on the population of vasoactive

intestinal peptide (VIP) ndash immunoreactive neurons in the porcine

small intestine

Katarzyna Palus1 Jarosław Całka

1 Barbara Jana

2


and Mazury in Olsztyn 2 Division of Reproductive Biology Institute of Animal Reproduction


Background

High levels of acrylamide have been

shown in food products manufactured and

processed at high temperatures (such as

chips cornflakes or coffee) [1] Although

the gastrointestinal tract is the main absorp-

tion route of acrylamide [2] little is known

about its effect on the enteric nervous

system (ENS) neurons The aim of present

study was to elucidate the impact of sup-

plementation of low and high doses of

acrylamide on the population of vasoactive

intestinal peptide (VIP)- immunoreactive

neurons in the porcine small intestine


The study was performed on 15 gilts

divided into 3 groups C group- the animals

were administered empty gelatine capsules

LD group- the animals were administrated

tolerable daily intake (TDI) dose (05 microgkg

bwday) of acrylamide capsules and HD

group- the animals were administrated high-

dose (5 microgkg bwday) acrylamide

capsules for 28 days After supplementation

period all animals were euthanized and

fragments of duodenum jejunum and ileum

were collected and fixed The frozen

sections (14microm thick) from the collected

intestines samples were then processed with

the double immunofluorescent staining

method using protein gene product 95 (PGP

95 mouse monoclonal Biogenesis cat

No 7863-2004 working dilution 11000

used here as a pan neuronal marker) and

VIP (rabbit polyclonal Biomol cat No

VA1285 working dilution 16000) antibody

as well as appropriate secondary antibody

(Alexa Fluor 488 and 546)

Results

Acrylamide supplementation affected

immunohistochemical characteristics of

ENS neurons in the porcine small intestine

The increase in the number of neurons

showing immunoreactivity towards VIP was

noted in all fragments studied The most

remarkable changes was noted in the inner

submucous plexus (ISP) in which a sta-

192

tistically important increase was observed in

both experimental group (LD and HD) in all

parts of intestine (duodenum from 1096 plusmn

065 in C group to 1421 plusmn 103 in LD

group and to 2065 plusmn 123 in HD group

jejunum from 1402 plusmn 064 to 2527 plusmn

094 and to 2992 plusmn 132 ileum from

1438 plusmn 098 to 1509 plusmn 091 and 2262

plusmn 152 respectively) In the outer

submucous plexus (OSP) statistically

important increase was noted in animals

receiving low and high doses of acrylamide

only in the ileum (from 1190 plusmn 029 to

1493 plusmn 026 and to 1545 plusmn 069 )

whereas in duodenum and jejunum only in

the HD group increase was important (from

1100 plusmn 008 to 1496 plusmn 045 and from

1454 plusmn 033 to 1847 plusmn 037) Similarly

in the myenteric plexus (MP) an increase in

number of neurons immunoreactive to VIP

was noted in both LD and HD group only in

ileum (from 1340 plusmn 138 in C group to

2068 plusmn 081 in LD group and to 287 plusmn

121 in HD group) In the duodenum and

jejunum in HD group an increase was also

significant (from 1214 plusmn 031 to 1671

plusmn163 and from 1170 plusmn 033 to 2014 plusmn

127)


The recorded changes revealed that even the

low doses of acrylamide influence the

nervous structures located in the porcine

small intestine wall This may result from

the neurotoxicity of acrylamide or from the

response of the ENS to the acrylamide-

induced inflammation and well corelate

with previous study confirmed neurotoxic

properties of acrylamide [3] VIP is known

to be an important neuroprotective factor

which stimulates mitosis within the

astrocytes supports neuronal differentiation

of embryonic stem cells and increases

neuronal survival under various patholo-

gical factors [4] Obtained results suggest

that VIP plays an important role in pro-

tecting the gastrointestinal tract during

acrylamide intoxication

References [1] F Van Lancker A Adams et al Chem Rev

111 (12) 7876-7903 2011 doi 101021

cr200032j

[2] B Zoumldl D Schmid et al Toxicology 232 (1-2)

99-108 2007

[3] RM Lo Pachin Toxicol In Vitro 25 573-579

2010

[4] K Makowska Neurogastroenterol Motil

30(11)e13439 2018 doi 101111nmo13439


Heat-induced changes in nuclear proteins associated with lamin

in the Drosophila melanogaster model system



1 Ryszard Rzepecki

1


Background









of heat-inducible genes and a global

downregulation of transcription Moreover

it has been found that after heat shock

induction the decondensation of chromatin

occurs [1]

Changes in interaction between chromatin and protein after heat shock were also observed with major karyoskeletal proteins involved in chromatin organizationndash lamin It belongs to V-type intermediate filaments

193

exerting structural and regulatory functions in the cell nucleus [2] Our hypothesis is that lamins together with topoisomerase II (Top2 is an enzyme required for DNA regulations) may play a key role in chromatin remodeling during HSR

For our studies we chose Drosophila mela-nogaster as a model system due to the presence of only two lamin genes ndash B-type (lam Dm) and A-type (lam C) and a single isoform of HSF which makes it a definitely simpler model than vertebrates In this study we focused on investigating diffe-rences between normal and heat shock condition with regard to changes in protein complexes associated with lamin Dm together with post-translational modify-cations which may be crucial in processes occurred during HSR

Material and Methods All experiments were performed on D melanogaster embryonic cell line ndash Kc Cells were maintained in suspension culture (in Schneider`s Drosophila Medium from Gibco with 10 FBS and 1 antibiotics) at 23degC as normal conditions To induce the heat shock cells were incubated at 37degC for 1 h before further experiments To identify proteins interacting with lamin 1 PFA cross-linking (10 min RT) followed by co-immunoprecipitation (co-IP) under dena-turing conditions (based on the protocol from ThermoFisher dedicated to Pierce Protein AG Magnetic Beads) Samples after co-IP were next digest by FASP method tryptic peptides were analyzed by tandem mass spectrometry analysis (LC-MSMS) MSMS data were processed using the Mascot searching engine (UniProt Drosophila database combined with The common Repository of Adventitious Proteins cRAP)

Results We aimed to confirm the interaction between lamin Dm and topoisomerase II in

both normal and heat shock conditions We observed extreme change in the number of proteins identified in MS after heat shock (almost 70 more interactors identified in comparison to control) After the classi-fication of identified proteins we observed changes in clusters in both groups based on protein functions In HS samples we observed an increased number of proteins involved in DNARNA binding Based on the quantitative analysis we showed about 30 decreased of lamC identifiers (the best-known interactor of lamDm q-value= 003) 30 increase of Top2 identification after hs (but the result is ns)




194

Life support system in a remote Mars colony

Paweł Piszko1 Natalia Ćwilichowska

2 Joanna Kuźma

3 Anna Jurga

4

12Faculty of Chemistry Wroclaw University of Science and Technology 3Faculty of Mechanical and Power Engineering Wroclaw University of Science and Technology 4Faculty of of Environmental Engineering Wroclaw University of Science and Technology

Background The concept of the Twardowsky Mars colony for 1000 people was granted second place in The Mars Colony Prize 2019 Further research on the colonyrsquos life support systems led to publishing a paper [1] which deepens and thoroughly analyses proposed solutions Mars harsh conditions lead causes multiple problems to encounter in terms of life support The reduced gravity lack of breathable atmosphere and insufficient recourses to name a few Based on the cutting-edge technology and space research there are numerous solutions to those problems In this study it is aimed to intro-duce a closed-loop system which could potentially sustain human presence on the Red Planet Four depended systems were introduced and described atmosphere system solid waste system water and wastewater system and biomass system

Material and Methods The conducted research was based on the up-to-date information regarding human organism behavior in extra-terrestrial condi-tions Additionally numerous analysis and simulations were conducted in scope of the waste management and daily dietary demand A variety of organic waste processing methods were investigated with respect to sup-porting 1000 inhabitants in environment of the planet Mars

Results As a result of research life support systems for remote mars colony has been proposed and published [1]

An atmosphere biomass solid waste water and wastewater systems was presented and their functionality analyzed in terms of life support in Martian conditions for 1000 people

Processing methods and specific solutions were described Mathematical simulations of the bioreactor flow and waste mana-gement were presented and calculated with redundancy

Discussion and conclusions The introduced concept gave a proof of technological feasibility and showcased a problem of subsystems implementation as a unity Moreover the emphasis (followed by calculations) was put closed loop of resources and efficiency of the proposed processes regarding life sustainment

There are certain research problems regar-ding the described systems that should be examined in the foreseeable future

atmosphere subsystem reducing the weight efficiency improvement reduction of energy demand

water subsystem inoculation of biolo-gical reactors the effect of reduced gravity on the aeration system and the collection of excessive sludge reco-very of elements from brine from physicochemical devices

solid waste subsystem significant improving the efficiency recovering of valuable chemical compounds and development of technology to process them

biomass subsystem clogging of aero-ponics precipitation in each system collecting harvests by the crew


[1] Ćwilichowska N Kuźma J Jurga A Piszko P amp Nguyen T (2019) Life support system in the mature Martian colony for 1000 people In E3S Web of Conferences (Vol 100) EDP Sciences httpsdoiorg101051e3sconf201910000013 [2] M S Anderson M K Ewert J F Keener Life Support Baseline Values and Assumptions

195

Document (National Aeronautics and Space Administration Washington DC Report No NASATP-2015ndash218570 2018) [3] D Bureau S Kaushik C Cho Bioenergetics (Fish Nutrition third ed Academic Press New York NY 1-50 2002) [4] D J Barta K D Pickering C Meyer S Pensinger L Vega M Flynn A Jackson

R Wheeler A Biologically-Based Alternative Water Processor for Long Duration Space Missions (NASA Johnson Space Center Houston TX United States Report No NASAJSC-CN-33488 2015) [5] P Zabel M Bamsey D Schubert M Tajmar Life Sci Space Res 10 1-16 (2016) 26

Fig1 Scheme of life support systems dependencies


Interaction of rhodium(III) cytostatic complex with red blood cells

and cell membranes

Hanna Pruchnik1 Adam Siadak

2 Aleksandra Włoch

1

1Faculty of Life Sciences and Technology Department of Physics and Biophysics Wrocław

University of Environmental and Life Sciences 2 Student of Faculty of Biology and Animal

Science Wrocław University of Environmental and Life Sciences

Background

The interest in the antitumor activity of

metal compounds was caused by B Rosen-

bergs discovery of the cytostatic effect of

cis-diaminodichloroplatin (II) Since then

many new metal complexes have been

studied for their potential use in medicine

Very promising cytostatic properties are

exhibited by Co Rh Ir and Pd coordination

compounds with nitrogen and phosphine

ligands [1 2] Our initialprevious tests for

the anti-tumor activity of the rhodium (III)

complex with tris (2-carboxyethyl) phos-

phine (in short ndash RhTCEP) have shown that

it is cytotoxic to many cell lines eg SK-

mel (malignant melanoma) SH-4 (mela-

notic melanoma) Colo-829 (malignant

melanoma) and C-32 (amelanotic melano-

196

ma) [work in preparation] To explain the

mechanism of biological activity of

RhTCEP a number of further studies should

be performed including interactions with

biological membranes ndash many chemical

compounds exhibit biological activity

through their direct or indirect effect on

protein structures and cell membranes

The overall purpose of the research task was

to investigate the effect of RhTCEP on red

blood cells and membranes This task has

been divided into the following stages

1 Examine the hemolytic activitytoxicity

of RhTCEP

2 Check the effect of RhTCEP on the

properties of the erythrocytersquos membrane

3 Check the influence of RhTCEP on the

structure and properties of lipid model

membranes (liposomes)


The analysis was conducted on erythrocytes

(porcine) erythrocyte lipid-protein mem-

branes (RBCM) and lipid membranes (lipo-

somes) without (SUV) and with cholesterol

(SUVchol) The research was carried out

using several complementary methods The

spectrometric method was used to perform

the first task The second task was

performed based on an optical microscope

(analysis of erythrocyte shapes) fluores-

cence spectroscopy with the use of fluo-

rescent probes as markers of lipid

membranes and spectroscopy of attenuated

total infrared reflection (Fourier transform

infrared attenuated total reflectance FTIR-

ATR) Fluorimetric method was also used to

measure fluorescence anisotropy and the

degree of ordering of lipid bilayers of the

model membrane (task 3) All measu-

rements were performed at 37 degC

Results Hemolysis is a process during which hemo-

globin flows out of the cell as a result of

damage to the membrane or an increase in

its permeability Tests conducted over

a wide range of concentrations from 10 to

200 M of the test compound showed that

RhTCEP does not cause membrane damage

It probably interacts with the hydrophilic

area of the outer monolayer of the mem-

brane as evidenced by the change in the

shape of 60 M RhTCEP modified blood

cells

Infrared spectroscopy provided a lot of

information about the structure and

intermolecular interactions in the protein-

lipid membrane Characteristic frequency

bands of the protein and lipid components

were analyzed in the IR spectra which can

be divided into the spectral regions that

originate from the molecular vibrations of

the hydrocarbon tail the interface region

and the head group We identified the

RBCM frequency bands for individual

membrane components methyl groups and

methylene hydrocarbon chains carbonyl

phosphate and choline groups of lipids and

also the amide I II and III bands The

RhTCEP does not change the fluidity of

RBCM in the hydrophobic region of the

lipid bilayer However slight changes are

visible in the vibration bands derived from

the phosphate group (νasPO2- and νsPO2

-) of

the polar part of the phospholipid mem-

branes The νasPO2- vibration band shows

high sensitivity to environmental polarity

and the possibility of interaction via

hydrogen bonds Changes in this area

indicate that RhTCEP interacts mainly with

the polar part of the lipid membrane This is

also evidenced by the results of measure-

ments of anisotropy and of generalized

polarization factor (GP)

Conclusions

In summary we can conclude that RhTCEP

in the studied concentration range is not

hemolytic active It does not disturb the

erythrocyte membrane whereas it slightly

affects its properties

197

References [1] M Gielen E R T Tiekink Eds Metallo-

therapeutic Drugs and Metal-Based Diagnostic

Agents The Use of Metals in Medicin Wiley

Chichester 2005

[2] H Pruchnik M Latocha et al Journal of

Organometallic Chemistry vol 822 74-79 2016

DOI 101016jjorganchem201608005

This work was sponsored by the statutory

activities of the Department of Physics and

Biophysics of Wrocław University of Envi-

ronmental and Life Sciences


Impact naringenin and its derivatives on p53 and Bcl2Bax expression

in human colon adenocarcinoma cells HT29

Oskar Przybyszewski1 Joanna Kozłowska

2 Mirosław Anioł

2 Dagmara Baczyńska

3

1Faculty of Pharmacy with Division of Laboratory Diagnostics Wroclaw Medical

University Borowska 211A 50-556 Wrocław Poland 2Department of Chemistry Wrocław

University of Environmental and Life Sciences Norwida 25 50-375 Wrocław Poland 3Department of Molecular and Cellular Biology Faculty of Pharmacy with Division of

Laboratory Diagnostics Wroclaw Medical University Borowska 211A 50-556 Wrocław

Poland

Background

Naringenin is a natural flavonon present in

many plants especially in citrus fruits It has

been shown as an potential anticancer agent

[1] However the molecular mechanism of

its action is still unclear Some studies indi-

cate that naringenin may induce an internal

pathway of apoptosis by regulation of p53

caspase 9 and Bcl-2 family members

expression [2] On the other hand its effect-

tiveness is limited by low stability and

relatively high IC50 vales For these reasons

new naringenin derivatives are being sought

to improve the anticancer therapy Previo-

usly our group has shown that 74-di-O-

butylnaringenin and its oxime have stronger

cytotoxic effect on human colon adeno-

carcinoma cell lines than naringenin [3]


We used HT29 colorectal adenocarcinoma

cells for our research First we induced

apoptosis by add Naryngenin and itrsquos

derivatives into the cells and then studied

the expression of p53 Blc-2 and Bax thanks

to Real-Time PCR and Western Blot

Furthermore we try to clarify the role of

apoptotic microRNAs such as miR125b

and miR155 in cellular death induced by

naringenin derivatives

Results

Our results indicate that all compounds

activate Bcl-2 and Bax expression but ratio

Bcl-2Bax only increases in HT29 cells after

treatment with naringenin and 74rsquo-di-O-

butylnaringenin In contrast ratio Bcl-2Bax

decreases following oxime 74rsquo-di-O-

butylnaringenin treatment and correlates

with its strong cytotoxic effect on cancer

cells On the other hand the oxime deri-

vative augments p53 expression at both

mRNA and protein level while naringenin

and its O-alkyl derivate do not affect TP53

gene expression


Our study demonstrates the molecular basis

for action of novel oxime derivative of

naringenin and provides it as a promising

agent in anticancer therapy

References

198

[1] Krauze-Baranowska M Marcinkowska K

Pobłocka-Olech L Naryngenina i jej pochodne

ndash flawanony o wielokierunkowej aktywności farma-

kologicznej Postępy Fitoterapii 2006 1 16-22

[2] Bazer FW Lim W Park S Song G

Naringenin-Induced Apoptotic Cell Death in

Prostate Cancer Cells Is Mediated via the

PI3KAKT and MAPK Signaling Pathways

J Cell Biochemistry 2017 118 (5) 1118-1131 doi

101002jcb25729

[3] Kozłowska J Grela E Baczyńska D

Grabowiecka A Anioł M Novel O-alkyl

derivatives of naringenin and their oximes with

antimicrobial and anticancer activity Molecules

2019 24 (4) art 679 doi 103390molecules

24040679


Sonoporation as a novel method for overcoming multi-drug resistance

phenomena in human gastrointestinal cancer cells


2 Julita Kulbacka

2



Background

Some types of cancers especially gastro-

intestinal cancers display resistance to the

chemotherapy which is called multi-drug

resistance (MDR) phenomenon Unfor-

tunately despite successful surgical treat-

ment the problem of the cancer cell

resistance to chemotherapeutics remains

unsolved resulting in the use of ever-higher

doses of cytostatic agents which often

prove to be ineffective Due to the nume-

rous clinical implications of the MDR

phenomenon there is a crucial need to seek

treatments that abolish or modulate MDR

One of them is called sonoporation ndash the use

of acoustic waves for temporary perme-

abilization of cell membranes which allows

maximization of targeted gene and drug

delivery to tumors while minimizing their

systemic toxicity The main aim of this

study was to investigate the effect of sono-

poration as well as sonoporation with

cytostatics calcium ions and microbubbles

and curcumin in tumor cells of the

gastrointestinal tract


As a research model we used the cells

sensitive and resistant to chemotherapy

from colon adenocarcinoma (LoVo LoVo

Dx) and pancreatic cancer (HPAF-II)

Preliminary studies included viability cell

death and cell cycle assays

Results and conclusions

Preliminary studies have shown that the

application of sonoporation in the presence

of nanobubbles increases the toxicity of

cytostatics The simultaneous use of sono-

poration bleomycin and nanobubbles

allowed for temporary permeabilization of

cell membranes and efficient drug pene-

tration into the cells It has been observed

that the effect of the therapy used depends

on the physical parameters of ultrasound

ie the power and pressure of the acoustic

wave A similar relationship was demon-

strated in the case of sonoporation with non-

toxic calcium chloride which caused

a significant decrease in cell survival Based

on these findings we claim that sonopo-

ration with appropriately selected parame-

ters and the appropriate concentration of

micro- and nanobubbles can increase the

therapeutic effect of drugs

References [1] I Lentacker I De Cock R Deckers S C De

Smedt and C T W Moonen Understanding

ultrasound induced sonoporation Definitions and

underlying mechanisms Adv Drug Deliv Rev

vol 72 pp 49-64 Jun 2014

199

Fig 1 Cellular effects induced by sonoporation [1]

Fig 2 Setup designed for the sonoporation of cells in suspension


Opposite effects of antioxidants on cancer and normal mammalian

cells exposed to subcosmic conditions during the stratospheric balloon

campaign


1 Julita Kulbacka

1


Background

The current age of dynamic development of

the space industry brings the mankind closer

to routine manned space flights and space

tourism This progress leads to a demand for

intensive astrobiological research aimed at

improving strategies of the pharmacological

protection of the human cells against extreme

conditions Although routine research in

space remains out of our reach it is worth

noticing that the unique severe environment

of the Earthrsquos stratosphere has been found to

mimic subcosmic conditions giving rise to

the opportunity to use the stratospheric sur-

face as a research model for the astrobio-

logical studies Our study included laun-

200

ching into the stratosphere a balloon

containing mammalian normal and cancer

cells treated with various compounds to

examine whether these substances can

protect the cells against stress caused by

rapidly varying temperature pressure and

radiation especially UV Due to oxidative

stress caused by irradiation and temperature

shock we used natural compounds which

display antioxidant properties namely

ndash catechin isolated from green tea honokiol

derived from magnolia curcumin from

turmeric and cinnamon extract After-

flight laboratory tests have shown the most

active antioxidants as potential agents which

can minimize harmful impact of extreme

conditions on human cells


Human ovarian cancer cells (SKOV-3

described as cancer cells) and non-cancer

Chinese hamster ovary cells (CHO-K1

described as normal cells) after 24-hour

incubation with various antioxidants were

detached suspended in freezing medium

Bambankertrade and placed in microtubes 30

minutes before the balloon flight Then the

samples were transported on ice to the

starting point and placed in a radiation

transmitting gondola located on the

environmental measurement unit with

accelerometer and temperature pressure and

UV sensors One half of the samples was

covered with aluminum foil to protect the

cells against irradiation ndash mostly UV

another half was sent into the stratosphere

without the protective layer As a result

we were able to evaluate the effect of

radiation on examined cells in the presence

of various antioxidants As a controls we

used not treated with antioxidants and not

sent into the stratosphere samples which

were incubated at 37degC 5 CO2 during the

flight Directly after landing biological


laboratory where after-flight tests were

performed (see Fig1)

Results The biological samples were launched to the stratosphere on the 30th of April 2018 from Wrocław Poland (51deg06236 N 17deg0332 E) At the highest altitude the temperature reached the lowest level of -35degC and the lowest pressure (1252 Pa) was measured Data provided by two UV sensors showed extreme exposure to the UV radiation causing immediate damage of unprotected human skin and eyes In the upper parts of the atmosphere the UV dose was more than twofold the dose correlating with the maximum dose in the UV-Index scale (reaching nearly 2463 mV)

Our work has led us to a conclusion that the application of the carefully selected com-pounds enables us to manipulate cellular stress response depending on the type of cells Final conclusions about the highest protective potential should be drawn based on the genotoxicity assays and cell death assays Altogether these findings suggest that honokiol and catechin have the best protective effect on the normal cells whereas curcumin and cinnamon act as radio- and light-sensitizers increasing the percentage of apoptotic cancer cells and DNA damage

Discussion and conclusions The results constitute a significant step towards the investigation of possible strategies for the cell protection in space environment and provide new insights into the application of the examined compounds for the prevention and treatment of cancer Due to its relatively low costs our approach remains the economic alternative for simulated subcosmic conditions conducted in the laboratory which require far more expensive specialized measurements

References [1] D Przystupski AGoacuterska et al Frontiers in Pharmacology vol 10 851 2020 DOI 103389fphar201900851

201

Fig 1 Procedure of experiment and balloon flight


The establishment of insulin resistance model in L6 cell

Joanna Pytel1 Agnieszka Chwiłkowska

2


Background Recent studies show that insulin resistance (IR) can precede the development of type 2 diabetes by several years It is described as the condition in which the sensitivity of peripheral tissues to insulin deteriorates As a result the pancreas produce more and more of this hormone the tissues become more resistant and glycaemic remains unstable Currently theres no specific insulin-sensitizing drug which could reverse IR symptoms

Muscle cells can be submitted to any treatment that may have an impact on insulin sensitivity and glucose uptake measurement will quantify this impact [1] The aim of this study was to demonstrate induced insulin resistance model

Material and Methods The experiment was conducted using rat skeletal muscle cell line L6 L6 myoblasts were induced to differentiation and myotube

formation There was assessed translocation of glucose transporter 4 (GLUT4) to plasma membrane by immunocytochemistry method The measurement of glucose uptake on cultured myotubes was done to evaluated impact of different conditions and medi-cines At the same time an MTT test was performed to assess the effect of variable conditions on cell survival

Results We showed that by stimulating L6 cells with high glucose and high insulin doses we successfully established in vitro IR model There was noticed the translocation of glucose transporter 4 (GLUT4) to plasma membrane

Discussion and conclusions The in vitro model is suitable for the test of any compounds that could improve insulin responsiveness or could prevent or reverse acquired or induced insulin resistance [2 3]

202

References [1] A Yap S Nishiumi et al Cytotechnology vol 55 103-108 2017 Doi 101007s10616-007-9107-y

[2] CJ Bailey S Turner Diabetes Obes Meta vol 6 293-8 2004 Doi101111j1462-89022004 00350x [3] S Chanon C Durand et al J Vis Exp vol 124 e55743 2017 Doi10379155743


Evaluation of tumor stem cells in LoVo HT29 and MCF7 cell lines in three-dimensional cell cultures

Anna Radajewska1 Oskar Przybyszewski

1 Helena Moreira

2 Ewa Barg

2

F Emhemmed3 C D Muller

3

1Student Research Group of Flow Cytometry and Biomedical Research at the Department of Basic Medical Sciences Wroclaw Medical University 2Department of Basic Medical Sciences Wroclaw Medical University 3IPHC UMR 7178 Univeriste de Strasbourg Illkirch France

Background Cancer stem cells (CSC) constitute a small cells population that due to their pluripotent properties are able to differentiate into any type of cancer cell Nowadays many studies emphasize a key role of CSC in the processes of tumor metastasis angiogenesis and resistance to cancer therapy which is important in the formation and evolution of tumors [1 2]The cells within the spheres can proliferate freely in every direction similar to in vivo conditions These 3D tumor cells due to the specific culture conditions should demonstrate more CSC characteristics comparted to 2D culture Increased expression of CD44 CD133 and CD326 antigens indicate the native nature of cancer cells growing in spheres

Material and Methods We used three-dimensional (3D) cell cultu-res due to the possibility of reproducing the most similar conditions of tumor growth by imitating tumor formation In our work we develop new 3D cultures so-called spheres based on special hydrophobic powder which separates the medium containing the cells from the environment The cells within the spheres can proliferate freely in every direction similar to in vivo conditions Afterwards we used flow cytometry

to indicate sought antigens (CD44 CD326 CD133)

Results Here we compare the expression level of antigens CD44 CD133 and CD326 in 3D cultures of colon and breast cancer cells sensitive and resistant to the cytotoxic drugs

Discussion and conclusions The development of new 3D cultures enriched in CSC population can be helpful in developing new therapeutic strategies for solid tumors In addition CSC analysis can contribute to a better understanding of the mechanisms of resistance to chemothe-rapeutic drugs and to the prevention of the chemoresistance and thus to the impro-vement of the effectiveness of currently used oncological therapies

References [1] M Szaryńska Z Kmieć The role of cancer stem cells in pathogenesis and therapy of cancer Forum Medycyny Rodzinnej czasopismo Polskiego Towarzystwa Medycyny Rodzinne j 1897-3590 T 5 nr 1 (2011) s 47-56 [2] K Wieczorek J Niewiarowska Cancer stem cells Postępy Higieny i Medycyny Doświadczalnej (Advances in Hygiene and Experimental Medicine) 66(855199)629-636 middot September 2012


203

Biological Properties of Dental Materials

Zbigniew Raszewski

RampD SpofaDental Czech Republic

Background and aim Dental materials belong to the medical devices which have a contact with human body in different time intervals (short period few minutes until few days) or long when they affect the human body for a long period of time ndash years The aim of this study was to compare cytotoxic properties of several dental materials on the cell cultures

Material and Methods There were verified six different products from SpofaDental portfolio were chosen for the study details are shown in Tab 1 Samp-les as a discs with thickens 1 mm and diameter 5 mm were prepared for tests For example alginate impression materials powder was mixed with water and set at room temperature Acrylic dental resins were used after mixing of powder (PMMA ) with liquid (MMA) Samples were tested for the cytotoxic properties by evaluation using the MTT (methyl thiazolyl dipheny-ltetrazolium bromide Sigma) on VERO CCL-81 (ATCC) cell line Cells were maintained in MEM medium containing 4 FBS in 37degC in humified atmosphere enriched by 5 CO2 Before exposition cells were suspended on 96-well plate in density 1x104 cells100 μl in MEMwell The cells were cultivated for 24 hours to obtained 80 of confluency and then were exposed to tested samples positive and negative controls after 24 hours Cell medium was removed and 6x 100 μl of the sample positive control negative control blank samples were added to individual wells The test article extract was prepared in 1x MEM cell growth medium (MEM supplemented with 10 fetal bovine serum extract) at the sample to extraction medium ratio of 60 cm2mL and extracted at 37 plusmn

1degC for 72 plusmn 2 hours The sample was unchanged by the extraction procedure and the extract was found to be clear and free of particulates After the incubation time with samples MTT test was applied using in final step 2-isopropanol (100 μglwell) with simultaneous shaking The absorbance was detected at 570 nm Based on the accep-tance criteria for the procedure there was judged that the viability of the cells is more than 70 after application of the test sample during 24 hours what is assumed that material has not cytotoxic properties

Table 1 Tested materials

Material Type of medical device

Elastic Cromo Class I contact time lt 5 min

FITT Class I contact time lt3-5 days

TAB2000 Class I contact time lt30 days

Adhesor Class IIa contact time long few years

Superacryl Plus Class IIa contact time 2-5 years

Mifam Super Lux Class IIa contact time 2-5 years

Results The obtained results are shown in Fig 1 The negative control (MEM) and blank sample (MEM with 4 FBS) both demonstra-ted no cytotoxic effect thus cell oxido-reductive potential was undisturbed The positive control (Sodium lauryl sulphate) demonstrated significant cytotoxic impact even after the shortest time of incubation (2h) The viability was less than 20 after 24 hours Additionally the cytotoxic effect of positive control has to be proven in all concentrations and negative control has to prove no cytotoxic potential The most

204

cytotoxic occurred Adhesor sample (ca 5) and the highest biosafety was observed for Mifam Superacryl and TAB 2000 (more than 70)

Conclusions In dental technology are using different kinds of materials Some of the has no cytotoxic properties (hot cured acrylic resins) Determination of cytotoxicity is one of the step in the development of new dental materials required normatively The results obtained allow the selection of appropriate raw materials When cytotoxicity is achie-

ved this does not completely cross out the product being developed as a Medical Device but requires further appropriate testing Because in the mouth materials can have a completely different effect than on isolated cell cultures Such example is material based on ZnO phosphoric acid which have been successfully used for over 150 years in dentistry (the first cement) Often the solvents (ethanol) contained in the material are responsible for the cytotoxic properties as in the product FITT

Figure 1 Cytotoxic properties of dental materials are varied and connected with the composition


Atorvastatin aided steroidogenic cells calcium electrochemotherapy

Nina Rembiałkowska1 Wojciech Szlasa

2 Karolina Cierluk


2

Anna Szewczyk1 Mounir Tarek

4 Jolanta Saczko

1 Julita Kulbacka

1


University 2Faculty of Medicine Wrocław Medical University 3Faculty of Chemistry


F-54000 Nancy

Background

Plasma membrane associated cholesterol

increases its resistance to electric pulse and

provides a proper environment for functio-

ning of membrane proteins some of which

like flotillin-2 play an important role in

cancer progression and survival [1] These

localise in cholesterol-rich structures called

rafts [2] This crucial role of cholesterol is

especially significant among steroidogenic

cells [3] The aim of this project is to

validate if the blockage of crucial for

cholesterol biosynthesis enzyme HMG-CoA

reductase can enhance the therapeutic effect

205

of microsecond calcium electrochemo-

therapy in comparison to control cells


MDA-MB231 (breast cancer) Du-145

(prostate cancer) cell lines were used as the

model of steroidogenic cells whereas A375

(melanoma) was a control cell line with

high cholesterol content in the membranes

but without the ability of steroid synthesis

To decrease the cholesterol biosynthesis the

cells were incubated for 2 days with ator-

vastatin Afterwards microsecond electro-

chemotherapy with calcium ions was perfor-

med (ESOPE protocol) The response to

therapy was compared with the cells that

have not been treated with the inhibitor The

viability of the cells was assessed by MTT

assay the permeability of the membranes

was studied with the use of flow cytometry

with PI The adhesive properties of the cells

were tested with scratch tests and immuno-

fluorescence studies Moreover the model

of increasing resistance of the cell mem-

brane with the increasing cholesterol

concentration was build with the use of

molecular dynamics simulations

Results

By including calcium ions in the

electroporation buffer the viability of the

cells could be significantly decreased

in comparison to the standalone electric

pulse Molecular dynamics studies indicate

that by the decrease in cholesterol content

the EP threshold decreases as well The

inhibition of the cholesterol biosynthesis

had a high impact on the steroidogenic cells

in comparison to the control cell line The

adhesive properties of the cells have also

changed in response to atorvastatin-aided

calcium electrochemotherapy


The decrease of cholesterol biosynthesis

could be used as an adjuvant in novel

chemotherapy approaches such as electro-

chemotherapy Even though the prelimi-

nary results are promising further studies

need to be done in this field in order to

apply this approach in clinical trials

References [1] X Wang et al Flotillin-2 is associated with

breast cancer progression and poor survival

outcomes J Transl Med vol 11 no 1 p 190

Aug 2013

[2] L J Pike Lipid rafts bringing order to chaos

J Lipid Res vol 44 no 4 pp 655-67 Apr 2003 [3] A Chimento et al Cholesterol and its

metabolites in tumor growth Therapeutic potential

of statins in cancer treatment Frontiers in Endocrinology vol 10 no JAN Frontiers Media

SA 2019

This research was financially supported by

the Ministry of Health subwention acoor-

ding to number of STMD26020142 from

the IT Simple system of Wroclaw Medical

University


Lipase-catalyzed synthesis of feruloylated lysophosphatidylcholine

Magdalena Rychlicka Natalia Niezgoda Anna Gliszczyńska

Department of Chemistry Wroclaw University of Environmental and Life Science Wroclaw

Poland

Background

The pro-health importance of phenolic acids

in human nutrition and disease prevention

has been well recognized and scientifically

confirmed One of the most abundant

phenolic acid with a wide array of thera-

peutic properties and lack of side known

effects is ferulic acid (FA) (4-hydroxy-3-

methoxycinnamic acid) In the resent years

the number of reports on the in vitro anti-

206

inflammatory antidiabetic anticarcino-

genic antimicrobial and hepato- cardio-

and neuroprotective [1] properties of ferulic

acid has increased significantly Despite

therapeutic effects FA is not use in industry

on the high scale The main reason is his

low bioavailability due to intensive transfor-

mation to the secondary metabolites in the

human body In studies on the rat model it

has been proven that the orally administered

free form of ferulic acid is already absorbed

from the stomach in 74 from where is

transferred to the liver via the portal vein

there is converted into sulfonic and glucuro-

nide derivatives which are excreted sub-

sequently via mainly urine [4] The result of

such rapid transformations is low content of

free form of ferulic acid in the general

circulation These data were also confirmed

in studies carried out on people Yang and

co-workers analyzed the pharmacokinetic

parameters of FA following administration

of oral FA (50 mg) as a sodium salt

(FA-Na) The maximum concentration of

free FA in the blood was only 25 μmolL

after 24 minutes and its half-life was only

42 minutes [5]

The latest research is therefore focused on

improving their physical and chemical

properties which would allow practical

application of the ferulic acid in the in-

dustry Promising in this respect are modi-

fications of the FA structure via covalent

bonding them with a lipid carrier especially

with phosphatidylcholine which is charac-

terized by high bioavailability in the human

body

The aim of our work was to develop of

a biotechnological method for the prepa-

ration of feruloylated phospholipids via

a one-step enzymatic reaction A novel

route for the synthesis of PC structured with

ferulic acid by enzymatic acidolysis and

interestrification of natural egg-yolk phos-

phatidylcholine was investigated Enzyme

screening effects of feruloyl donors (FA

and EF) and reaction variables (organic

solvent enzyme load reaction time and

substrate ratio) were also evaluated in the

process of production of FA-enriched PLs


Ferulic acid was subjected to lipophilization

with a natural phosphatidylcholine isolated

from egg-yolk by enzymatic acidolysis

interesterification in an organic solvent

environment For this purpose at the

beginning of the experiments commercially

available lipases were tested for their

activity to catalyze the regioselective

incorporation of phenolic acid residues into

the sn-1 position of natural phosphate-

dylcholine Next organic solvent and effects

of acyl donors (FA and EF) were evaluated

Then the response surface methodology

with 3 factors at 3 levels substrates molar

ratio enzyme loading and time of reaction

wre used for optimization of reaction of

interesterification of PC with EF Modified

phospholipids were purified and analyzed

by means of chromatographic methods

(SPE column chromatography TLC GC

HPLC) The structure of obtained product

was confirmed by spectroscopic data

Results We investigated and optimized the process of one-step enzymatic synthesis of struc-tured phosphatidylcholine (PC) ewith ferulic acid (FA) Four different immobile-zed lipases were evaluated as biocatalysts for this purpose Novozym 435 and a binary solvent system of toluenechloroform 91 (vv) were found to be the most suitable biocatalyst and medium respectively which significantly increased the incorporation of FA into PLs fraction The selected opti-mized parameters were set as 115 molar ratio PCEF 30 (ww) of the enzyme load and 6 days of incubation time The maximal experimental incorporation of FA into phospholipid fraction (PCLPC) was 18 mol The process of interesterification of egg-yolk PC with EF catalyzed by Novo-

207

zym 435 at the optimized parameters carried out in the large scale afforded feruloylated lysophosphatidylcholine (FLPC) in high 62 isolated yield This is the first study reporting the preparation of FLPC by one-step enzymatic method which is a promi-sing in the context of the production of food supplements and additives based on the phenolic acids and natural PC

Discussion and conclusions A novel biotechnological route of incorpo-ration of ferulic acid (FA) into phospho-lipids was successfully developed Presented method is then promising in the area of enzymatic production of phospholipid bio-

preparation containing biologically active ferulic acid (18 of incorporation) with potential application in the food industry as food ingredients natural emulsifiers or nu-traceuticals with proved pro-health activity

References [1] Z Zhao M Moghadasian et al Food Chemistry 109(4) 691-702 2008 DOI 101016jfoodchem200802039 [2] Z Zhao Y Egashira The Journal of Nutrition 134(11) 1136-1142 2004 DOI DOI101093 jn134113083 [3] C Yang Y Tian et al Journal of Pharma ceutical and Biomedical Analysis 43 945-950 2007 DOI101016jjpba200609027


Expression of neuronal isoform of nitric oxide synthase (nNOS)

in the porcine enteric neurons of jejunum following prolonged

indomethacin supplementation

Dominika Rząp1 Barbara Jana

2 Jarosław Całka

1

1Faculty of Veterinary Medicine University of Warmia and Mazury in Olsztyn 2Division of Reproductive Biology Institute of Animal Reproduction and Food Research Polish Academy of Sciences Olsztyn

Background The enteric nervous system (ENS) also called the second brain consists of a mesh-like system of neurons and is capable of acting independently of the sympathetic and parasympathetic nervous systems The ENS outside the central nervous system is one of the main regulators of gastrointestinal func-tioning and contributes to tissue response to the various pathological conditions [1]

Indomethacin is a nonsteroidal anti-inflammatory drug (NSAID) commonly used to reduce fever pain stiffness and swelling from inflammation

The goal of the present study was to deter-mine the influence of high doses of indo-methacin on the neurons in the enteric nervous system of the porcine jejunum It should be noticed that selection of the drug active substance and animal species

model were not accidental Swine is consi-dered to be one of the main type of animals in biomedical researches

Material and Methods The experiment was performed on 8 imma-ture gilts of the Pietrain x Duroc breed weighting approximately 20 kg and around 8 weeks old The animals were divided into 2 groups ndash control (n=4) and experimental (n=4) The gilts constituting control group received empty gelatin capsules orally while pigs from the study group were given indomethacin orally 10 mgkg of body weight The experiment lasted 4 weeks then pigs were euthanized After fixation and freezing section double immunofluores-cence staining was performed Antibodies against the protein gene-product 95 (PGP 95) neuronal marker and against the nNOS were used as primary antibodies while Alexa Fluor 488 and 546 were used as

208

secondary antibodies Stained 14 μm sections were examined under Olympus BX51 fluorescence microscope

Results In the present study supplementation with indomethacin caused changes in the neuro-chemical phenotype of nerve cells Analysis of the obtained results showed that inflam-mation caused by long-term administration of high doses of indomethacin resulted in decrease of the number of nNOS positive neurons in the myenteric and submucosal ganglia in the porcine jejunum

Discussion and conclusions The present immunohistochemical studies revealed that in the jejunum of pigs treated with high doses of indomethacin subpo-pulations of nNOS-immunoreactive myenteric and submucous neurons were statistically altered Previous studies have also shown similar changes in this neuroactive sub-stance in the swine gastrointestinal tract during induced inflammatory processes [2 3] It is believed that the capacity to change neurotransmitterneuropeptide content termed enteric neuroplasticity is an adap-tation to an unfavorable enteric micro-environment [4]

Changes of nNOS expression in enteric neurons suggest the neurochemical adapta-tion of these neurons to the conditions of induced inflammation and possible invol-vement in local repair processes

References [1] Furness JB Callaghan BP et al The enteric nervous system and gastrointestinal innervation integrated local and central control Adv Exp Med Biol vol 817 39-71 2014 doi 101007978-1-4939-0897-4_3 [2] Szymańska K Całka J et al Nitric oxide as an active substance in the enteric neurons of the porcine digestive tract in physiological conditions and under intoxication with bisphenol A (BPA) Nitric Oxide vol 80 1-11 2018 doi 101016 jniox201808001 [3] Burliński PJ Rychlik A et al Effects of inflammation and axotomy on expression of acetylcholine transferase and nitric oxide synthetase within the cocaine- and amphetamine-regulated transcript-immunoreactive neurons of the porcine descending colon J Comp Pathol vol 150 (2-3) 287-96 2014 doi 101016jjcpa201308007 [4] Schaumlfer KH Van Ginneken C et al Plasticity and neural stem cells in the enteric nervous system Anat Rec (Hoboken) vol 292(12) 1940-52 2009 doi 101002ar21033



Osteoprotegerin in carotid atherosclerosis ndash preliminary research

Agnieszka Sapa-Wojciechowska1 Alina Rak-Pasikowska

1 Paweł Gliński

2 Kornel

Pormańczuk34

Iwona Bil-Lula1

1Division of Clinical Chemistry and Laboratory Hematology Department of Medical Laboratory Diagnostics Faculty of Pharmacy Wroclaw Medical University 2Department of Laboratory Diagnostics Lower Silesian Oncology Center in Wroclaw 3Department of Surgery 4th Military Teaching Hospital in Wroclaw 4Division of Surgical Specialties Department of Clinical Nursing Faculty of Health Science Wroclaw Medical University

Background Carotid artery stenosis is a main reason of stroke and cognitive dysfunction Plaque instability is responsible for acute cardio-vascular events One of the elements of plaque vulnerability is calcification which

is regulated via osteoprotegerin (OPG) receptor activator of NF-κB ligand (RANKL)receptor activator of NF-κB (RANK) system and OPG is a candidate biomarker of atherosclerosis

209


plasma concentration of OPG in patients

with carotid stenosis and its correlation with

routine laboratory test results We also

aimed to evaluate the OPG tissue content in

parts of artery with atherosclerosis


20 patients after routine carotid endarte-

rectomy at the Department of Vascular

Surgery of 4th Military Teaching Hospital

in Wroclaw were enrolled in this study The

OPG concentration was measured in

part of removed artery with visible calci-

fied and ulcerative changes ndash plaque

part of removed artery without visible

symptoms of atherosclerotic lesions ndash

reference tissue

citrated plasma collected before surgery

The OPG concentration was determined by

ELISA Prior to analysis tissue fragments

were homogenized in liquid nitrogen and

then lysed with homogenization buffer and

Pellet Pestlereg Motor The laboratory results

were routinely performed before surgery in

a hospital laboratory The statistical analysis

was performed in GraphPad Prism 5

Results

We observed higher amount of osteo-

protegerin in reference tissue than in the

area of the plaque (p = 00009) (Figure 1)

There was no correlation between plasma

OPG and laboratory test results as presented

in Table 1

Table 1 Correlation between routine test

results and OPG in plasma

Parameter Correlation Parameter Correlation

Total

cholesterol

r = 02791

p = 02335 Triglycerides

r = 009936

p = 06768

LDL-

cholesterol

r = 0225

p = 03403

White blood

count

r = -03164

p = 01741

HDL-

cholesterol

r = 01734

p = 04647

Platelet

count

r = 03544

p = 01252

Non-HDL-

cholesterol

r = 02143

p = 03644 Creatinine

r = -01964

p = 04066


Strong calcification in the core of the plaque

may be a result of smaller amount of OPG

because OPG is an inhibitor of vascular

calcification [12] Hakimi et al showed that

OPG was the highest in the marginal part of

the plaque (assessed by immunohisto-

chemical staining) [3] We suppose that

lower amount of OPG in the middle part of

the lesion may be also a result of OPG

degradation by proteases released from

inflammatory cells massively infiltrating the

plaque OPG has been extensively investi-

gated and its diagnostic potential as a marker

of aortic plaque presence or plaque vulne-

rability is well documented however

osteoprotegerin has not been included in

routine use so far and further research is

required [2 4]

References


example below

[1] L Rochette A Meloux et al Calcif Tissue Int

vol 105 239-51 2019 DOI 101007s00223-019-

00573-6

[2] F Montecucco S Steffens et al Clin Dev

Immunol 200775805 DOI 101155200775805

[3] M Hakimi A Hyhlik-Duumlrr et al Int J Mol

Med vol 31 331-8 2013 DOI 103892

ijmm20131401

[4] M Abedin T Omland et al Am J Cardiol vol

99 513-8 2007 DOI 101016jamjcard

200608064

210

Figure 1 Comparison of the content of OPG in tested tissue fragments


Determination of methanol ethanol and isopropanol in the gas phase

using IR spectroscopy

Przemysław Skibiński1 Dariusz Sarzyński

1 Olimpia Gładysz

1

1Department of Analytical Chemistry Wroclaw Medical University Wroclaw Poland

Background

The The aim of this work was to develop an

analytical method to detect methanol

ethanol and isopropanol in the gas phase

using IR spectroscopy The procedure

involves recording the Fourier-transform

infrared spectroscopy (FTIR) spectra of

alcohols in gas phases and also determi-

nation of analytical wave numbers and

plotting the dependence of the absorbance

on the pressure of the analysed component

The reliability of measurements was checked

in the presence of other gaseous compo-

nents ie nitrogen air


Measurements were carried out in a cell

filled with a gaseous form of the tested

alcohols in a vacuum apparatus under

controlled pressure to 1middot10-5 atm

Results

The results of the research allowed to

determine several analytical wave numbers

useful in quantitative measurements of the

tested compounds in the pressure range

from 65middot10-4 atm to 13middot10-1 atm (27middot 10-5

moldm3 to 54middot10-3 moldm3) The calib-

ration curves were characterized by high

linearity It was found that quantitative

measurements of alcohols are also possible

in the presence of other gases eg nitrogen

air and in a mixture of alcohols


Alcohols used in the work are the most

commonly used solvents for the extraction

of plant materials Although FTIR is a

technique that is rarely used for the

quantification of residual solvents [1] it can

be proposed for rapid initial evaluation of

the residues of some solvents used

in pharmaceutical procedures

References [1] Grodowska K Parczewski A Analytical

methods for residual solvents determination in

pharmaceutical products Acta poloniae pharma-

ceutica 67 13-26 2010


211

Morphological restructuring of the optic nerve in the early stages of

streptozotocin-induced diabetes mellitus in chronic stress

Skora J S1 Shchur M B

2

1Faculty of Foreign citizensrsquo training department Ivano-Frankivsk national medical university Ukraine 2Doctor ndash ophthalmologist

Background Diabetes mellitus (DM) makes up 40-70 of the total population in various countries of the world and ranks first among other endocrine diseases [4 5] Medico-social problem of DM is caused by early disability and mortality of patients due to specific complications ndash microangiopathy (nephro-pathy retinopathy) macroangiopathy (myo-cardial infarction stroke lower extremity gangrene neuropathies [3-5] Therefore the purpose of our research was to study the ultrastructural rearrangement of the optic nerve of rats in streptozotocin diabetes mellitus (SDM) under chronic stress

Material and Methods 20 adult white male rats (weighing 180-200 g) were used for the study All animals were divided into 4 groups 1 group with modeled SDM and chronic immobilization stress 2 group ndash animals with SDM 3 group ndash with chronic immobilization stress 4 group ndash intact rats The SDM was simulated by a single intra-peritoneal administration of streptozotocin SIGMA (USA) (6 mg per 100 g of body weight) which was diluted in 01 M citrate buffer with pH 45 Chronic immobilization stress was performed by placing the animal in a sealed plastic container 5 hours per day The material was collected 14 days after the beginning of the SDM simulation using histological electron microscopic bioche-mical and statistical methods

Results On the 14th day of experiment blood glucose and HbA1c level of 1st group of rats were the highest in comparison to group 4 and were 1552plusmn124 mmoll (plt0001) and

708plusmn043 (plt001) respec-tively in the 2nd group ndash 1412plusmn103 mmoll (plt0001) and 613plusmn053 (plt001) in the 3rd group ndash 512plusmn056 mmoll (pgt005) and 227plusmn032 (pgt005) in the 4th group of animals the above indicators were 443plusmn063 mmoll and 206plusmn025 Such biochemical changes in the 1st and 2nd groups of rats indicate the development of experimental DM of mode-rate severity On the background of hyper-glycemia in the 1 and 2 study groups in comparison to intact rats there was a spasm of arterioles which was confirmed by a probable decrease in their lumen area by 15-23 times (plt001) with the Wogen-worth index increasing by 14-21 times (plt005) indicating a decrease in their capacity According to the morpho-metric analysis the area of the myelin nerve fibers (MNF) was significantly increased in 13-18 times (plt005) as a result of an increased area of the myelin sheath by 153-187 (p lt005) which indicates the edema of the latter In comparison with intact indi-cators in the MNF of the 1st and 2nd groups of rats the index g decreases to 016plusmn005 and 017plusmn003 (intact indicators ndash 055plusmn003 p=00012) which also indi-cates the edema of the myelin sheath In the 3rd group of rats probable morphometric changes of the MNF were not detected At the ultrastruc-tural level in the rats of group 1 and 2 we have discovered the stratification of myelin lamella in MNF peryaxonal swelling disorganization of mitochondrial cristae and enlightenment of their matrix in axoplazm of axial cylinders Individual MNF with signs of anisochromia partial easing and destruction of the myelin sheath are identified At the same time a significant

212

portion of MNF with saved structure is noticeable

Discussion and conclusions It should be noted that in SDM and its combination with chronic immobilization stress firstly we have observed the changes in the hemomic-rocirculatory blood flow of the intracranial part of the optic nerve which manifested by the spasm of arteriols which is associated with elevated level of contrinsular hormones [2] Disturbances of blood microcirculation create conditions for demyelination of MNF which is confirmed with the results of our morphometric study The changes which we have identified in the MNF are interpreted by other authors as a violation of axonal flow in axial cylinders [1 3] which occurs as a result of swelling of the myeline sheth and can be interpreted as periaxonal degeneration According to the data [3] the disturbance of carbohydrate metabolism is associated with a deficiency of glycosaminoglycans which promotes rapid degenerative-dystrophic change in the peripheral nerves Even in the absence of clinical signs morphological and histoche-mical examination reveals a reduced number of macro-microvessels and changes in their wall occurrence of which we associate with the reaction of morphological

elements of Schwann cells and with the violation of hemodynamics of the nerve

Thus in the early stages of the development of SDM there is a violation of the blood supply to the optic nerve which leads to the destruction of the myelin sheath of the MNF In this case chronic immobilization stress exacerbates the course of the disease

References [1] Herashchenko SB Dieltsova OI Kolomiitsev AK Chaikovskyi YuB Peryferiinyi nerv (neiro-sudynno-desmalni vzaiemovidnoshennia v normi ta patolohii) Ternopil Ukrmedknyha 2005 342 s [2] Tkachuk YuL Morfo-funktsionalni zminy hipofizarno-nadnyrkovoi systemy u ranni terminy rozvytku eksperymentalnoho tsukrovoho diabetu Svit medytsyny ta biolohii 20151 159-162 [3] Khachaiants NIu Dyabetycheskaia polyneiro-patyia Uspekhy sovremennoho estestvoznanyia 2015 3 87-924 [4] Kocdor P Kaya S Erdil M Cureoglu S Paparella MM Adams ME Vascular and Neuro-epithelial Histopathology of the Saccule in Humans With Diabetes Mellitus Otol Neurotol 2016 Jun 37(5) 553-7 doi 101097MAO0000000000001018 5 Ponto KA Koenig J Peto T Lamparter J Raum P Wild PS et al Prevalence of diabetic retinopathy in screening-detected diabetes mellitus results from the Gutenberg Health Study (GHS) Diabetologia 2016 59(9) 191-193


Structural rearrangement of the endocrine system of the heart

in the early stages of diabetes mellitus in conditions of chronic stress

Skora J S1 Vasyliuk V M

2 Vlasiuk T I

3 Zhurakivska O V

4 Zhurakivska O Ya

5

Dutchak U M6

1Faculty of Foreign citizensrsquo training department Ivano-Frankivsk national medical university Ukraine 2Department of Clinical Anatomy and Operative Surgery Ivano-Frankivsk national medical university Ukraine 356Department of Human Anatomy Ivano-Frankivsk national medical university Ukraine 4Medical faculty Ivano-Frankivsk national medical university Ukraine

Background The medical and biological problem of dia-betes mellitus (DM) is predetermined by its high prevalence and development of severe

complications that can lead to disability or even death [1] Many people are currently under constant stress Although an adaptive syndrome has got a protective function in

213

some cases the response of the organism may not be adequate to the conditions that caused it Moreover chronic stress leads to angina heart attack stroke which are more commonly diagnosed and have a more difficult course in patients with DM [2] Therefore the purpose of our research was to study the ultrastructural rearrangement of secretory atrial cardiomyocytes (SAC) in streptozotocin-induced diabetes mellitus rat model (SDM) under chronic stress


20 adult white male Vistar rats (180-200 g

of body mass) were used for the research

All animals were equally divided into 4

groups (5 animals in each group) 1 group

of animals with modeled SDM and chronic

immobilization stress 2 group ndash with SDM

3 group ndash with chronic immobilization stress

4 group ndash intact animals The SDM was

simulated by a single intraperitoneal admini-

stration of streptozotocin SIGMA (USA)

which was diluted in 01 M citrate buffer

with pH 45 (6 mg per 100 g of body

weight) Chronic immobilization stress was

performed by placing the animal in a sealed

plastic container 5 hours per day during 14

days The pieces of auricles of the heart

were collected 14 days after the SDM

simulation Histological method (hemato-

xylin eosin) transmission electron micros-

copy biochemical (blood glucose glycated

hemoglobin ANP) and nonparametric

statistical methods were used

Results

On the 14th day of the experiment the levels

of glucose and HbA1c in the blood of 1st

group of rats were the highest in compa-

rison with group 4 and were respectively

1521plusmn133 mmoll (p lt0001) and 778plusmn058

(plt001) in group 2 ndash 1372plusmn153 mmoll

(plt0001) and 608plusmn045 (plt001) in

group 3 - 521plusmn038 mmoll (pgt005) and

208plusmn017 (pgt 005) In the 4th group of

animals the above indicators were 485plusmn063

mmoll and 178plusmn018 Such biochemical

changes in the 1st and 2nd groups of rats

indicate the development of decomposed

DM On the background of hyperglycemia

in the 1st and 2nd study groups there was an

increase in the morphofunctional activity of

SAC which was confirmed by hypertrophy

and hyperplasia of the Golgi complex

extension of cisternae of granular endo-

plasmic reticulum increase in the bulk

density of secretory granules (SG) of SAC

in 23-15 times in comparison with intact

indicators due to all their forms especially

young and diffusing This indicates enhan-

ced processes of synthesis and excretion of

atrial natriuretic peptide (ANP) from the

cell The level of the latter in the blood

increases in 26 times in the 1st group and in

18 times in the 2nd group In animals of

group 3 we observed a decrease in the bulk

density of SG in 08 times due to the

decrease in their young and mature forms

while the bulk density of diffusing SG was

significantly increased but the level of ANP

in the blood did not differ significantly from

those in the intact group of animals


According to various authors the develop-

ment of SDM in rats leads to an increased

activity of the pituitary-adrenal system with

hyperproduction of adrenocorticotropic

hormone and hypercortisolemia [3] Accor-

ding to our studies such metabolic changes

in bodies of rats of 1st and 2nd groups with

SDM lead to an increased functional acti-

vity of the endocrine system of the heart

Yes We have found a significant increase

in the bulk density of the SG due to all their

forms with a predominance of diffusing

which leads to an increase of ANP Such

changes occur due to the following patho-

genetic mechanisms mechanical stretching

of the myocardium due to an increase in

circulating blood volume which occured as

a result of an increased polyol pathway of

glucose metabolism [4] polydipsia [5 6]

214

vascular spasm due to elevated levels of

contrinsular hormones [3]

Thus in the early stages of the development

of SDM there is an increase in the functio-

nal activity of SAC which is confirmed by

an increase in the bulk density of the SG

due to all their forms especially diffusing

and indicates the enhanced processes of

synthesis and secretion of ANP during this

period of the experiment and the increase of

its level in the blood In this case chronic

immobilization stress exacerbates the disease

References [1] Madonna R Balistreri CR et all Diabetic

microangiopathy Pathogenetic insights and novel

therapeutic approaches Vascul Pharmacol 2017

90 1-7 doi 101016jvph201701004

[2] Francesco Cosentino Peter J Grant Victor

Aboyans 2019 ESC guidelines on diabetes pre-

diabetes and cardiovascular diseases developed in

collaboration with the EASD European Heart

Journal 2019 1-69

[3] Tkachuk YuL Morfo-funktsionalni zminy

hipofizarno-nadnyrkovoi systemy u ranni terminy

rozvytku eksperymentalnoho tsukrovoho diabetu

Svit medytsyny ta biolohii 20151 159-162

[4] Kovaacutets T Tomcsaacutenyi J Bradycardia and B-type

natriuretic peptide Int J Cardiol 2008 3 137

[5] Shlapak IP Halushko OA Intensyvna terapiia

diabetychnoho ketoatsydozu u khvorykh na ishe-

michnyi insult Ostrыe y neotlozhnыe sostoianyia

v praktyke vracha 20151 812

[6] Benedini S Villa P Luzi L Bevilacqua M

Pioglitazone does not modify ANP levels of type 2

diabetic patients World Journal of Cardiovascular

Diseases 2012 2 277-282


Psychophysiology of empathy ndash galvanic skin reaction

Michał Skorupski1 Bartłomiej Mazur

1 Bożena Gulla

1

1Faculty of Applied Psychology Jagiellonian University

Background

The term empathy was first used by

Titchener in 1909 [1] and has been present

in colloquial language literature and art

ever since Despite deep cultural rootedness

both a widely recognized definition and

effective explanation of its biological

foundations are lacking [2] The first person

to conduct the research on empathy in the

context of psychophysiology using galvanic

skin reaction as the main measurement was

probably Dennis Krebbs who published his

results in 1975 [3] Although the author

managed to combine the strength of the

momentary empathic response with an

increase in GSR readings the methodology

he used is controversial because he comp-

letely omitted empathy-trait as a variable

Several modern studies confirm the exis-

ence of a relationship between the skin

galvanic response to emotive stimuli and the

level of empathy [4 while others reject this

thesis [5] The inconsistency of the results

of previous studies leaves the question about

the possibility of linking GSR with empathy

open Answering it would open up new

possibilities in objectifying the measu-

rement of empathy


The measurement of empathy was con-

ducted using the Polish adaptation of Baron-

Cohensrsquo EQ-short scale (SSIE) [6] and the

Empathic Sensitivity Scale (SWE) [7]

Galvanic skin reaction was measured using

a galvanometer included in the Stoelting

polygraph

The empathic stimuli used in the research

were taken from Affective Picture List [8]

and Nencki Affective Word List [9] and

additionally checked in a pre-test

A total of 65 students were examined in the

study

215

The study received a positive opinion of the

Scientific Research Ethics Committee of the

Institute of Applied Psychology Jagiello-

nian University

Results

GSR levels were found to be the highest

when subjects were presented with negative

emotive stimuli (plt0001) and lowest when

the stimuli was positive (p=0011) A total

of 15 analyses were conducted to check the

relationship between each of the scales of

the two empathy questionnaires and galva-

nic-skin reaction to each type of the stimuli

As a result of multiple regression analysis it

was found that the general SWE result is a

predictor of the strength of skin-galvanic

reaction strength to negative stimuli

(r = 054 p = 002) and the general SSIE

result is a negative predictor of skin-

galvanic reaction strength to positive stimuli

(r = -041 plt001) The remaining 13

relationships were found to be statistically

insignificant (pgt 005)


Although it was proven that different types

of emotive stimuli evoke different levels of

galvanic-skin reaction the authors were

unable to link the strength of the GSR

to empathy levels

References [1] Titchener E (1909) Lectures on the

experimental psychology of the thought- processes

Nowy Jork The MacMillan Company

[2] Batson C (2009) These Things Called Empthy

Eight Related but Distinct Phenomena [W]

Decety J Ickes W (red) The Social

Neuroscience of Empathy (s 3- 16) Cambridge

The MIT Press

[4] Bogdanov V Bogdanova O (2013)

Alexythymia and empathy predict changes in

autonomical arousal during affective stimulation

Cognitive Behavioral Neurology 26 (3) s 121-132

[5] Oliveira-Silva P Gonccedilalves Oacute (2011)

Responding Empathically A Question of Heart not

a Question of Skin Applied Psychophysiology and

Biofeedback 36(3) s 201-207

[6] Jankowiak-Siuda K Siemieniuk K

Grabowska A (2009) Neurobiologiczne podstawy

empatii Neuropsychiatria i Neuropsychologia t

4(2) 51-58

[7] Kaźmierczak M Plopa M Retowski S

(2007) Skala wrażliwości empatycznej Przegląd

Psychologiczny 50(1) 9-24

[8] Lang P Bradley M Cuthbert B (2008)

International affective picture system (IAPS)

Affective ratings of pictures and instruction manual

Technical Report A-8 University of Florida

Gainesville FL

[9] Riegel M Wierzba M Wypych M Żurawski

Ł Jednoroacuteg K Grabowska A Marchewka A

(2015) Nencki Affective Word List (NAWL) the

cultural adaptation of the Berlin Affective Word List

ndash Reloaded (BAWL-R) Behavior Research

Methods 47 (4)


Influence of fluoride on endocrine tissue

Marta Skoacuterka-Majewicz1

Wojciech Żwierełło1 Daniel Styburski

1 Patrycja

Kapczuk2 Justyna Kikut


3 Natalia Komorniak

3 Joanna

Palma3 Nina Konecka

4

1Department of Medical Chemistry Pomeranian Medical University in Szczecin

Powstańcoacutew Wlkp 71 Str 70-111 Szczecin Poland 2Department of Biochemistry

Pomeranian Medical University in Szczecin Powstańcoacutew Wlkp 72 St 70-111 Szczecin

Poland 3Department of Human Nutrition and Metabolomics Pomeranian Medical

University in Szczecin Broniewskiego 24 Str 71-460 Szczecin Poland 4Depertament Of

Neurocognitive Science Pomeranian Medical University in Szczecin Unii Lubelskiej 1 Str

Szczecin 71-252 Poland

216

Environment pollution is a serious problem

for developed and developing countries

Anthropogenic pollution results in the

constant emission of hazardous substances

that contaminate water air and soil leading

to such problems as global warming and

smog but also ndash the increase of the concen-

tration of heavy metals and toxic elements

including fluorine Dutkiewicz placed

fluorine on the list of top 5 of the most

dangerous environmental toxins as early as

in 1995 Therefore it is important to inves-

tigate and trace substances which contribute

to the development of disorders in the

human body

The first observations suggest that fluoride

has a negative influence on the functioning

of the thyroid ovaries and testicles The

current state of knowledge suggests a

significant effect of this element on the

decrease in sex hormone levels which may

in turn cause impairment of fertility and

puberty Most studies confirm that sodium

fluoride causes an increase in TSH concen-

tration and a decrease in the concentration

of T3 and T4 secreted by the thyroid glands

In addition there were correlations between

NaF and an increase in parathyroid hormone

secretion without significant effects on body

calcium It is possible that fluoride adver-

sely affects the amount of insulin leading to

impaired pancreatic function resulting in

impaired glucose tolerance A decrease in

cortisol secreted by the adrenal glands was

also observed

There are observations that indicate fluoride

its toxic influence on the endocrine system

but so far this phenomenon has not been

documented in detail The correlation

between potential inflammation within these

organs and the amount of hormones

released to the bloodstream seems to be

particularly interesting In recent years you

can see an increase in the incidence

of autoimmune diseases including Hashi-

motos disease Despite limited research no

undeniable pathway for endocrine toxicity

has been found thats why we should look

for the reasons for these upward trends


Nanoemulsions in the treatment of the vaginal infections

Michał Smoleński1 Katarzyna Małolepsza-Jarmołowska

1


Background

Infections of the female reproductive system

are still a challenge for modern medicine

Oral drug administration forces the use of

increased doses It has a negative influence

on patientsrsquo health increases the risk of

complications and superinfections caused

by a different microbial agent Intravaginal

route of drug administration provides higher

bioavailability due to rich vascularization

and avoiding first past effect through the

liver and intestine However individually

and temporally changing the composition

of vaginal mucus are the limiting factors of

the possibility of intravaginal drug admi-

nistration

Nanoemulsions are a thermodynamically unstable colloidal dispersion commonly consisting of oil in water droplets sized lt300 nm They are preferable over thermo-dynamically stable microemulsions due to their kinetic stability which is less affected by the change of conditions eg during the administration of the drug to the patient [12] Nanoemulsions present a high degree of adhesion to the mucosa and simultane-ously allow delivery of several active pharmaceutical ingredients (APIs) of diffe-rent hydrophilic or lipophilic properties

217

which makes them potential drug form for intravaginal administration

The aim of this study is determining the current state of knowledge in the intrava-ginal application of nanoemulsions in the treatment of female reproductive tract infections

Material and Methods Relevant papers were searched in the Web of Science and Scopus databases The scope of the research has been limited to records published in years 2014-2019 Different forms of words vaginal intravaginal emul-sion nanoemulsion were used as the keywords

Results Among the reviewed papers two main types of formulations can be distinguished ndash the nanoemulsions and emulgels based on nanoemulsions and two groups of consti-tuent activity ndash antifungal and antibacterial

Natural lipid fractions such as cholesterol oleic acid and soybean oil were used as an oil phase as well as synthetic like Labrafacreg Lipophile

As an API in the treatment in vaginal candidiasis oxiconazole nitrate clotrima-zole Syngonanthus nitens extract and Mentha spicata L var viridis aromatic oil was used Polyphenon 60 ciprofloxacin cranberry and curcumin were selected as an antibacterial agent

In addition to zeta potential droplet sized of every obtained nanoemulsion were charac-terized by dynamic light scattering methods Particle size varied from 23 nm to approx 300 nm Most of the researchers provided SEM or TEM images and rheological measurements were performed In vitro drug release tests were varied between groups ndash Franz diffusion cells dialysis bags and USP dissolution test apparatus II were used In some cases construction of ternary

phase diagrams was used in the techno-logical part of the research Only in a few articles information about the pH range of the formulation was provided ranging approx from 3 to 6 [3-9]

Discussion and conclusions Nanoemulsions enhance the effect of simultaneous administration of hydrophilic and lipophilic APIs Based on in vitro and in vivo studies natural-origin and synthetic antimicrobial compounds showed promising results in the treatment of both bacterial and fungal vaginal infections Most of the formulations present modified release profile Yet further studies are needed

It would be favourable to prepare analytical guidelines for the research on intravaginal nanoemulsion as there are not many requirements in European Pharmacopeia for these formulations There are significant differences the between analytical methods used to determine the properties of intra-vaginal nanoemulsions

References [1] D J McClements Soft Matter vol 8 6 1719-1729 2012 [2] N Anton T F Vandamme Pharmaceutical Research vol 28 5 978-985 2011 [3] A Kaur YSaxena et al AAPS PharmSciTech vol 18 6 2188-2202 2017 [4] M A dos Santos Ramos P Da Silva et al Journal of Biomedical Nanotechnology vol 15 5 1072-1089 2019 [5] J L Soriano-Ruiz A Calpena-Capmany et al International Journal of Pharmaceutics vol 554 105-115 2019 [6] N Srivastava D K Patel et al Journal of Drug Delivery Science and Technology vol 48 490ndash498 2018

[7] A Kaur S Gupta et al Advanced Pharma-ceutical Bulletin vol 7 4 611-619 2017 [8] K Atinderpal N Kapoor et al Indian Journal of Pharmaceutical Sciences vol 80 3 442-452 2018 [9] A Khattab S Ismail Indian Journal of Pharma-ceutical Sciences vol 8 3 33-40 2016


218

Biogenic and pyrogenic SiO2 nanoparticles ndash which of them are safer

for endothelial cells

Solarska-Ściuk K1 Adach K

2 Glatzel-Plucińska N

3 Olbromski M

3 Bonarska-

Kujawa D1 Fijałkowski M

2



Advanced Technologies and Innovation Technical University of Liberec Czech Republic 3Department of Histology and Embryology Medical University of Wrocław Wrocław

Poland

Background

Silica is a key component of almost every

species of plants and animals Silicon dioxide

in the form of nanoparticles posssesses

some unique physico-chemical properties

which make these nanoparticles a platform

useful in biology and medicine Moreover

due to their size shape and large surface to

volume ratio these nanoparticles are the

most promising material used as an

adsorbent filler and additive to drugs and

cosmetics [1] Furthermore due to their

biocompatibility the silca nanoparticles can

also be use as biosensors [2] biomarkers

and drug carriers [3] In this paper we

compared the biological effects of both

silica nanoparticles extracted from Urtica

dioica L and pyrogenic ie commercially

available silica nanoparticles


Biogenic and pyrogenic silica nanoparticles

were studied using SEM and TEM The

studies were conducted immortalized human

microvascular endothelial cells (HMEC-1)

The cells were cultured in MCDB 131

medium under 5 CO2 in plastic flasks at

37ordmC

The cytotoxic effects of NPs on cells were

determined after exposure to different

concentrations (0-200 microgml) at 24 48 and

72 h The cell viability was measured using

fluorimetric (Hoechst 33258) assay The

ability of the compounds cell cycle arrest

was studied using flow cytometry analysis

The cells were stained using FxCycle

PIRNase Staining Solution

Results

The study which was conducted using

transmission electron microscopy (TEM)

and scanning electron microscopy (SEM)

confirmed that the size of tested silica is

between 8 and 20 nanometers as well as its

amorphous structure In terms of chemical

composition we report that the nanopar-

ticles obtained by green chemistry method

(bioSiO2) have similar composition to

synthetically produced (pyrSiO2)

The obtained data indicated that SiO2 NPs

extracted from stinging nettle show higher

toxicity than pyrogenic NPs in immortalized

human microvascular endothelial cells

(HMEC-1) Furthermore the level of cyto-

toxicity is time and concentration depen-

dent In the current study we confirmed that

the exposition on pyrSiO2 did not elicite

statistically significant cell cycle arrest at

the G2M cell cycle phase

Another effect was observed after treatment

of cells with bioSiO2 nanoparticles Collected

data indicated on decreasing level of cells in

the G2M cell cycle phase This may

suggest that nanoparticles caused excessive

oxidative stress (which was confirmed in

previous study) and they are toxic for

HMEC-1 cells

219


In terms of chemical composition we found

that the particles obtained from an extracted

form of Urtica dioica L have similar com-

position to synthetically produced silica

Nanoparticles of biogenic silica obtained

from plant material are considered to be

a potential source of nanomaterial usable for

many applications Silica nanoparticles can

cause oxidative stress leading to DNA

damage cell cycle arrest and apoptosis

[45] What is more toxicity of silica

nanoparticles depends on type of nanopar-

ticles their size dose and cell type [6]

In addition the results proved that both

biogenic and synthetically produced nano-

partciles are safe for endothelial cells in

appropriate concentration (50 microgml) and

show good biocompatibility which makes

them promising candidates for future

studies

References [1] Hoet PHM Brueske-Hohlfeld I et al

J Nanotoxicol 2 1-2 2004 DOI org101186

1477-3155-2-12

[2] Zhang FF Wan Q et al Anal Bioanal Chem

380 637-642 2004

[3] Hirsch LR Stafford RJ et al Proc Natl

Acad Sci U S A 100 13549-13554 2003

[4] Asweto C O Wu J Int J Environ Res Public

Health 14289 2017 DOIorg103390ijerph

14030289

[5] Duan J Yu Y et al PLoS One Apr 19 62087

2013 DOI 101371journalpone006208

[6] Kim IY Joachim E et al Nanomedicine

Nanotechnology Biology and Medicine 11 1407-

1416 2015 DOIorg101016jnano201503004

Acknowledgements

This work was supported by grant for the

development of young scientists no

B030003219 and from funds of the


Physics and Biophysics Wroclaw Univer-

sity of Environmental and Life Sciences



Variability of serum immunoglobulin G degree of galactosylation

in women with endometriosis ndash pilot study

Katarzyna Sołkiewicz1 Hubert Krotkiewski

2 Marcin Jędryka

34 Ewa Maria Kratz

1


of Pharmacy Wroclaw Medical University Poland 2Institute of Immunology

and Experimental Therapy of the Polish Academy of Sciences Wroclaw Poland 3Department of Oncology Gynecological Oncology Clinic Faculty of Medicine Wroclaw

Medical University Poland 4Department of Gynecological Oncology Lower Silesian

Cancer Centre Poland

Background

Endometriosis is an inflammatory disease

characterized by the presence of endo-

metrial tissue outside of the uterine cavity

changed in its architecture and has been

associated with a wide range of factors

Diagnosis of endometriosis is solely made

through surgerylaparoscopy as does not

exist consistent biomarkers for disease

diagnostics [1] Immunoglobulin G (IgG) is

a main human serum protein glycoprotein

which is a powerful effector molecule that

can mediate tissue inflammation by comple-

ment activation The agalactosylated form

of IgG act as proinflammatory factor and

the increased expression of agalactosylated

forms of IgG is observed in some inflam-

matory diseases eg in rheumatoid arthritis

[2]

220

The aim of our study was the analysis of

serum IgG degree of galactosylationagalac-

tosylation in women suffering from endo-

metriosis


The serum samples ndash study group ndash were

collected at the Department of Gynae-

cological Oncology in Lower Silesian

Cancer Centre (Poland) A healthy control

group consisted of serum samples from

women without endometriosis The samples

were divided into two groups the control

group (n=10) and the group with endo-

metriosis (n=28) The degree of IgG

galactosylation was determined using a mo-

dified solid phase enzyme-linked immuno-

sorbent assay lectin-ELISA The method is

based on the relative reactivity of IgG

glycans with specific biotinylated lectins

Ricinus communis agglutinin I (RCAI)

ndash detect the terminal galactose Griffonia

simplicifolia II (GSL-II) ndash detect the

terminal GlcNAc To assess the galacto-

sylation status of IgG N-glycanrsquos the

agalactosylation factor (GSL-IIRCA-I) was

calculated [2] Statistical analysis was

performed using STATISTICA 133 PL

(StatSoft Inc) software (U Mann-Whitney

test)

Results

The results of statistical analysis show

significant differences in the relative reac-

tivity of IgG oligosaccharides with lectins

used between the control group (healthy

women) and the group of women suffering

from endometriosis (p lt 0005) The

agalactosylation factor was significantly

higher in group of patients with endo-

metriosis than in group of healthy women

(p lt 0005)


Due to the fact that IgG effector functions

are controlled by N-glycosylation its altered

galactosylation and agalactosylation status

can contribute to immune dysregulation in

chronic inflammatory diseases such as endo-

metriosis [3] The ratio of GSL-IIRCA-I

relative reactivity calculated for serum IgG

glycans may become an additional diagno-

stic marker of endometriosis

References [1] Greene AD Lang SA et al Endometriosis

where are we and where are we going

Reproduction 2016152(3)63-78

[2] Pasek M Duk M et al Galactosylation of IgG

from rheumatoid arthritis (RA) patients-changes

during therapy Glycoconj J 200623(7-8)463-71

[3] Reily CStewart TJ et al Glycosylation in

health and disease Nat Rev Nephrol 201915(6)

346-366


Quantum chemistry aided examination of antioxidative potential

of phenolic acids

Maciej Spiegel1 Beata Żbikowska

1 Zbigniew Sroka

1 Jerzy Hładyszowski

2

1Department of Pharmacognosy Faculty of Pharmacy Wroclaw Medical University 2Department of Physical Chemistry Faculty of Pharmacy Wroclaw Medical University

Background

Phenolic acids belong to the group of

polyphenolic compounds known for their

health-promoting effects resulting from the

antiradical activity [1]

The very main determinant of polyphenolsrsquo

scavenging potential is a presence of

hydroxyl groups linked with the aromatic

ring their substitution pattern and additional

residues [2]

221

In this study antiradical activity of phenolic

acids was investigated and new activity

index was proposed Experimental results

and computational quantum chemistry data

were statistically corelated

Table 1 Structures of investigated phenolic

acids

Phenolic acid 1 2 3 4 5 6

gentisic COOH O

H

H H OH H

homogentisic CH2COO

H

O

H

H H OH H

homoprotocat

echuic

CH2COO

H

H OH OH H H

p-coumaric CH=CHC

OOH

H H OH H H

4-

hydroxyphen

ylacetic

CH2COO

H

H H OH H H

-resorcylic COOH O

H

H OH H H

ferulic CH=CHC

OOH

H OC

H3

OH H H

homovanillic CH2COO

H

H OC

H3

OH H H

caffeic CH=CHC

OOH

H OH OH H H

syringic COOH H OC

H3

OH OC

H3

H

4-

hydorxybenz

oic

COOH H H OH H H

gallic COOH H OH OH OH H

m-coumaric CH=CHC

OOH

H OH H H H

protocatechui

c

COOH H OH OH H H

vanillic COOH H OC

H3

OH H H

-resorcylic COOH H OH H OH H

3-

hydroxybenz

oic

COOH H OH H H H

o-coumaric CH=CHC

OOH

O

H

H H H H

23-

dihydroxyben

COOH O

H

OH H H H

zoic

salicylic COOH O

H

H H H H

sinapic CH=CHC

OOH

H OC

H3

OH OC

H3

H

veratric COOH H OC

H3

OC

H3

H H


Investigated phenolic acids (see Table 1)

were tested with FRAP ABTS and DPPH

methods

Quantum chemistry pathway consisted of

obtaining isomers with the simulated

annealing and geometry optimization linked

with frequency computations up to

UB3LYP6-31G(dp) DFT method [3]

Elaboration of the hydrogen abstraction was

performed by successive removal of a

hydrogen atom from each hydroxyl or

methoxy group and repeating DFT calcula-

tions In order to obtain the reorganisation

energy (RE) energies of unrelaxed radicals

were noted The polarizable continuum

model (PCM) of the solvents was applied as

well [4]

The hydrogen abstraction energy (HAE)

describing energy required to remove

hydrogen atom from the given hydroxyl

group can be ascribed to the equation

Lai et al [5] transition state energy

determining the hydrogen abstraction rate is

stated to be

To check whether radical reorganisation

energy (REr) has an influence on hydrogen

abstraction kinetics we introduced HAIndex

Its main goal is to describe the transition

state energy up to some constant dependent

only on the hydrogen acceptor molecule

Thus the differences between HAIndex

values should correspond only to the

differences in transition state energies of

222

phenolic acids reacting with the same

abstractor in the same reaction medium

HAIndex can be described as

Results

The strongest activity reducing ion Fe3+ to

Fe2+ was noted for 23-dihydroxybenzoic

acid A slightly lower activity was observed

for homoprotocatechuic acid and gentisic

acid Similar results were obtained for

ABTS and DPPH tests The model of

hydroxylation by two hydroxyl group

situated next to a carboxyl group seems to

be the most effective alike observation can

be made for mutual ortho position model of

25-hydroxylation and hydroxyl groups

immediate vicinity to carboxyl residue


The results of quantum studies are well

correlated to the experimental data ndash Spear-

manrsquos rank order correlation coefficient

shows strongly negative correlation with a

two-tailed p-values less than 001 in most

cases

In conclusion it can be stated that

Compounds with only methoxy or one

hydroxyl group exhibit very low antioxidant

activity

Position C4 seems to be very important for

the antioxidative activity of phenols

Partial methoxylation of cinnamic and

benzoic acids at C3 and C5 positions and

hydroxylation at C4 promote antioxidant

activity

References [1] A Parr G Bolwell J Sci Food Agric vol 80

985-1012 2000 101002(sici)1097-0010

(20000515)807lt985aid-jsfa572gt33co2-z

[2] C Rice-Evans N Miller et al Free Radic Biol

Med vol 20 933-956 1996 1010160891-

5849(95)02227-9

[3] R G Parr Horizons of Quantum Chemistry vol

58 5-15 1980 101007978-94-009-9027-2_2

[4] M Cossi V Barone et al Chem Phys Lett vol

255327-33519961010160009-2614(96)00349-1

[5] Lai W Chunsen L et al Angew Chem Int Ed

vol 51 5556-5578 2012 101002anie201108398


Influence of Structural Features on the Antiradical Activity

of Flavones and Flavonols mdash A Quantum Chemical Study

Maciej Spiegel1 Tadeusz Andrunioacutew

2 Zbigniew Sroka

1

1Department of Pharmacognosy and Herbal Medicines Wroclaw Medical University

Borowska 211A 50ndash556 Wroclaw Poland 2Advanced Materials Engineering and Modeling

Group Wroclaw University of Science and Technology M Smoluchowskiego 23 50-372

Wroclaw Poland

Background

Flavones and flavonols are representatives

of a widespread group of dietary poly-

phenols which beneficial activity results

from the radical scavenging potential [1]

Presented studies were conducted in order

to examine the influence of the structural

features on these natural antioxidantsrsquo

hydrogen donating ability described here

by the enthalpy values of the first step of the

proposed mechanisms of action


The low-energy geometries of 13 investi-

gated compounds were generated using

molecular dynamics simulations Electronic

structure studies were performed starting

with the preoptimization at HF3-21G(d)

level of theory up to B3LYP6-31+G(dp)

223

in a water solvent using Polarizable Conti-

nuum Model [2 3]

Results

Computational data provided valuable

information about the electronic structure of

the investigated compounds Each aspect

was deeply investigated and discussed as

follows


Natural Bond Orbital analysis demonstrated

significant influence of py orbitals conju-

gation on the systemrsquos energy reduction

manifested in the mutual planarity of rings

B and C Moreover the contribution of the

C2=C3 double bond and carbonyl residue in

an electron flow among investigated flavor-

noidsrsquo backbone has been elucidated

Thermochemical calculations indicated that

the most prominent mechanism of hydrogen

abstraction is Sequential Proton Loss

Electron Transfer from C7 hydroxyl group

Consideration of intramolecular hydrogen

bonds is remarkably important while

describing the formation of the radical

understood as a hydrogen-donating ability

ndash their presence could lower enthalpy up to

7 kcalmol Sroka et al in their experimental

studies have shown over a hundredfold

difference between the measured activity of

apigenin and luteolin [4] Theoretical studies

somehow clarified this unusual behavior

with an existence of catechol group in

luteolin structure which undergoes two

reactions forming stable o-hydroquinone

structure in the end Appliance of Frontier

Molecular Orbital Theory and visualization

of the obtained results allowed to refer the

electron density distribution to the hyper-

conjugation effect and the chemical

hardness The mechanism of intramolecular

hydrogen swap between C5 hydroxyl group

and the carbonyl oxygen of the radical was

proposed and investigated ndash though it could

extend delocalization the studies didnrsquot

prove it is likely to occur

References [1] G R Beecher Journal of Nutrition vol 133(10)

3248S-3254S 2003 101093jn133103248S

[2] J Tomasi B Mennucci et al Chemical

Reviews vol 105(8) 2999-3094 2005 101021

cr9904009

[3] A D Becke The Journal of Chemical Physics

vol 98(7) 5648-5652 1993 1010631464913

[4] Z Sroka B Żbikowska et al Journal

of Molecular Modeling vol 21(12) 307 2015

101007s00894-015-2848-1


Spectrophotometric analysis of creatinine in artificial and mixed urine

with increased sarcosine content

Martina Staňkovaacute1 Michaela Všetičkovaacute

1 Dagmar Uhliacuteřovaacute

1 Zuzana Toacutethovaacute

1

Branislav Ruttkay-Nedeckyacute12

Josef Růžička1 Rene Kizek

12

1Department of Research and Development Prevention Medicals sro Tovaacuterniacute 342 742 13

Studeacutenka-Butovice Czech Republic 2Department of Human Pharmacology and Toxicology

VFU Brno Palackeacuteho třiacuteda 19461 612 42 Brno Czech Republic

Background

Creatinine (CREA) is produced in muscles

by irreversible non-enzymatic dehydration

and spontaneous cyclization from creatine

and (after phosphate cleavage) from creatine

phosphate Creatine phosphate serves as

a source of energy in muscle CREA cannot

be re-phosphorylated and passes into the

blood and is excreted in the urine In the

body CREA is formed at a relatively cons-

tant rate Its formation is related to muscle

mass and is stable under normal calm and

224

meatless diet (with normal glomerular

filtration) It is very often monitored to

assess renal function but CREA levels have

also been monitored in relation to various

cancers (ovaria cancer head and neck

cancer hepatocellular breast cancer etc 1-4)

For the determination of CREA the Jaffe

reaction is used to react with picrate in an

alkaline medium to form a red-orange

complex In this method other components

of biological fluids (pyruvate acetate and

others) react with picrate New tumor

markers are intensively searched for rapid

diagnosis One potential tumor marker of

prostate cancer could be the amino acid

sarcosine (SAR) Increased concentrations

of SAR were found in the urine of patients

with prostate cancer The aim of this study

was to detect CREA in various urine types

in the presence of SAR


Mindray BS300 analyzer was used for the

analyzes The chemicals used for the

analysis were purchased from Merck

Artificial urine was prepared according to

published protocols Mixed urine was

prepared by mixing urine of the volunteers

(n = 5) Distilled water was prepared by the

Aqual system and the ultrapure water was

prepared by the ELGA system up to 18 MΩ

water quality The determination of pH was

performed on a pH meter Concentrations of

ions were determined by ISE (K Na Cl)

SAR was analyzed by Trinder reaction on a

plate photometer for 30 min All analyzed

data were transferred to the QUINSLAB

laboratory database and statistically

evaluated


Analysis of CREA is a routine and long-

term method in clinical biochemistry

However slight variations in determination

of CREA concentration are not an obstacle

to the interpretation of the results The

ability to monitor CREA and possibly

another analyte present in the urine may

require the determination of the CREA

concentration as accurately as possible

There were three main blocks of analyzes in

our work A) dependence of absorbance

on CREA concentration in different matri-

ces B) dependence of absorbance on CREA

concentration in different matrices in the

presence of SAR (25 micromolL) C) changes

in analytical SAR response (25 micromolL)

in different matrices and CREA concen-

trations (0-100 mmolL) Thus n = 60

replicates of individual CREA concen-

trations were analyzed The mean linear

dependence was described by the equation

y = 0091x + 00405 (r = 09960) QC 1276

RSD 1383 Within the range of lower

creatinine concentrations (0-10 mmolL) the

dependence was linear (r = 09955) RSD

1262 QC 1241 LOD (083) and

LOQ (275) mmolL We found that CREA

detection is similar in all studied matrices

In the presence of SAR no effect on the

CREA signal was also observed and

correlation analysis was -SAR + SAR with

r = 0999 When monitoring the SAR signal

as a function of increasing concentration (0-

100 mmolL) of CREA slight changes in

trend (r = 02-03) were observed in AU-N

(artificial urine) trend changes (r = 03-04)

were also observed for AU-7 (artificial

urine) and the downward trend in mixed

urine 11 was most pronounced (r = 055-

058) When evaluating the data obtained in

the control diagram 90 of the values

were in the 1SD band

Conclusions

Detection of CREA by the proposed

procedure was reproducible in all types of

samples analyzed No changes in CREA

detection were observed in the presence of

SAR CREA (30 mmolL) interfered

slightly with SAR and a decrease in signal

was observed

225

Acknowledgements The work was carried out with the support of Liga proti rakovine Praha 2732019

References [1] G Nishimura M Tsukuda et al Int J Clin Oncol vol 12 120-124 doi101007s10147-006-0635-9 (2007)

[2] M J Montgomery P M Beringer et al Ther Drug Monit vol 22 695-700 doi101097 00007691-200012000-00008 (2000) [3] J L Limquiaco G L H Wong et al J Gastroenterol Hepatol vol 24 63-69 doi101111 j1440-1746200805701x (2009) [4] R Kent V P Gopalakrishnan et al Kidney Int vol 91 761-762 doi101016jkint201608004 (2017)


Anti-inflammatory and skin regenerating properties of vegetable oils

Aneta Starzec1 Malwina Brożyna

2 Łukasz Kotyra

3 Izabela Fecka

1

1Faculty of Pharmacognosy and Herbal Medicines Wroclaw Medical University 2Faculty Pharmaceutical Microbiology and Parasitology Wroclaw Medical University 3Faculty of Medical Biochemistry Wroclaw Medical University

Skin is the largest organ of the body and is a barrier between external and internal-environment Every day skin is exposed to many harmful factors such as mechanical damage pathogens ultraviolet radiation (UV) allergens and irritants Skin damage and lack of proper skin care result in inflammatory processes which can lead to the development of chronic inflammatory skin diseases eg atopic dermatitis

Plant oils are natural fats extracted from different parts of plants (seeds fruits or sprouts) Oils depending on the place of origin and chemical composition have different beneficial effects on the condition of the epidermal barrier reducing TELW (transepidermal water loss) creating occlu-sive layers restoring structures of stratum corneum and participating in biological processes such as hormone synthesis cell division inflammatory processes and metabolism of lipids and amino acids

The aim of this paper is to present a few selected vegetable oils (coconut oil pome-granate seed oil rose hip oil Calophyllum inophyllum oil argan oil) that may be useful in the production of cosmetic and pharma-ceutical emulsion with anti-inflammatory and skin regenerating properties

References [1] L Tzu-Kai L Zhong et al International Journal of Molecular Sciences 19 70 1-21 2018 doi103390ijms19010070 [2] SR Varma TO Sivaprakasam et al Journal of Traditional and Complementary Medicine XXX 1-10 2017 doi101016jjtcme201706012 [3] U Avsar Z Halici et al Ostomy Wound Manage 63 3 26-34 2016 [4] T Leacuteguillier M Lecsouml-Bornet et al PLoS One 10 9 2015 doi 101371journalpone0138602 [5] T K Karagounis J K Gittler et al Pediatric Dermatology 36 9-15 2019 DOI 101111 pde13621 [6] T Aburjai FM Natesheh Phytotherapy Research 17 987-1000 2003 DOI 101002

ptr1363


Contamination of the API with nitrosamines

A Starzyńska1 S Stasiak

1 A Trukan

1 E Sawicka

2

1Studentsrsquo Scientific Society at the Department of Toxicology Faculty Pharmacy Wroclaw Medical University Wroclaw Poland 2Department of Toxicology Faculty Pharmacy Wroclaw Medical University Wroclaw Poland

226

Drug manufacturers have been obligated by

the European Medicines Agency (EMA) to

carry out a risk analysis of nitrosamines

contamination in produced medicines Over

the past two years a significant amount

of nitrosamines has been detected in specific

batches of drugs such as valsartan raniti-

dine or recently metformin

It has been shown that the contamination

source of valsartan preparations a popular

antihypertensive drug could be starting

materials reagents and solvents used in the

synthesis of the API (Active Pharmaceutical

Ingredient) Valsartan contains a tetrazole

ring The synthesis of this ring requires the

use of sodium nitrite which reacts with

amine or trace amine solvents to form

nitrosamines The risk of cancer has been

investigated with patients taking conta-

minated valsartan preparations

The latest information shows that N-nitro-

sodimethylamine (NDMA) impurities have

also been found in metformin preparations

which may be the result of a technological

process Metformin remains the first-line

medication controlling high blood sugar in

patients with type II diabetes and its abrupt

withdrawal may result in health conse-

quences

Interesting results have been shown in

studies on preparations of ranitidine ndash an H2

receptor antagonist which is one of the

most commonly used pharmaceuticals in the

treatment of gastroesophageal reflux disease

and peptic ulcers Urinary NDMA excretion

after clinically used doses of ranitidine was

evaluated Researchers confirmed the pro-

duction of N-nitrosodimethylamine (NDMA)

a potent carcinogen by nitrosating raniti-

dine at gastric pH conditions The work is

a review of the latest publications on this

important topic Observations made by

scientists suggest the need for risk asses-

sment related to NDMA toxicity the concen-

tration of which can increase significantly in

the body with chronic use of these drugs

Therefore it is extremely important to use

alternative treatment methods that minimize

exposure to N-nitrosamines

References [1] httpswwwwhointmedicinespublicationsd

rugalertsInformationNote_Nitrosamine-

impuritiesen

[2] A Pottegaringrd K Kristensen M Ernst N

Johansen P Quartarolo J Hallas Use of

N-nitrosodimethylamine (NDMA) contaminated

valsartan products and risk of cancer Danish

nationwide cohort study BMJ 2018 362 doi

httpsdoiorg101136bmjk3851


Petunidin-3-O-glucoside as a natural antioxidant which is in interaction

with the model membrane human albumin and plasmid DNA

Paulina Strugała1 Klaudia Dzida

2 Teresa Kral

13 Martin Hof

3 Janina Gabrielska

1


Sciences Wrocław Poland 2OrgChem Student Scientific Circle at the Department

of Chemistry Wrocław University of Environmental and Life Sciences Wrocław Poland 3Department of Biophysical Chemistry J Heyrovsky Institute of Physical Chemistry

of the Czech Academy of Sciences Prague Czech Republic

Background Anthocyanins are natural dyes which are

present in plants as derivatives of -O-gly-coside with the most popular substituent ie

glucose and main aglycones including cyanidin delphinidin petunidin malvidin pelargonidin and peonidin [1] Conducted studies indicate on great importance of anthocyanins as prondashhealth food ingre-

227

dients Such an opinion is based on many biological activities which are shown by these compounds in particular antioxidant anti-inflammatory anticancer cardiopro-tective activities as well as their role in vision improvement protection against diabetics and against degeneration of neurons [2 3]

The aim of this research was to investigate antioxidant properties of petunidin-3-O-glu-coside (Pt-3-glu) as well as to determine the consequences of its interaction with biomo-lecules ie mimic lipid membrane which reflects the lipid membrane composition of tumor cells human serum albumin and plasmid DNA

Material and Methods Antioxidant activity Pt-3-glu was investi-gated using fluorometric method in refe-rence to mimic lipid membrane in which free radicals were induced in a way of thermal decomposition of AAPH Using fluorescence probes DPH and MC540 which were located at different depths of a lipid bilayer the impact of Pt-3-glu on the properties of both hydrophobic and hydrophilic areas within mimic membranes was determined Determination of binding between Pt-3-glu and albumin was carried out in a way of fluorescence quenching of albumin and using steady state fluorescence spectroscopy Interaction of plasmid DNA molecules with Pt-3-glu was performed using fluorescence correlation spectroscopy with the function of single photon counting (TCSPC-FCS) enabling tracking of dyna-mics changes at the level of single mole-cules while tracking changes in fluores-cence lifetime

Results The conducted studies indicated that Pt-3-glu shows high antioxidant activity (IC50=244plusmn024 microM) as well as it protects lipids against the peroxidation process more effectively than ascorbic acid

(IC50=12944plusmn1243 microM) Studies based on the use of fluorescent probes showed that Pt-3-glu is mainly localized on hydrophilic part of bilayer and is responsible for increasing rigidification of this part of a membrane Such a location of Pt-3-glu in hydrophilic part of a membrane may be a sort of a barrier which protects a mem-brane against the detrimental impact of free radicals In addition stiffening effect of lipid molecules may impact on limitation of free radicals diffusion in a membrane In studies focused on interactions with the main blood protein it was indicated that Pt-3-glu is able to quench natural fluorescence of albumin which is bond with this com-pound as a result of an impact of hydrogen bonds and van der Waals forces The measurement results using TCSPC-FCS method showed that Pt-3-glu actively interacts with plasmid DNA and causes its condensation

Conclusions On the basis of performed research and observed biological activity of a tested anthocyanin namely petunidin-3-O-glucoside it is concluded that this compound may be used in both pharmaceutical and food industry

Acknowledgments The research was financed by the (Polish) National Science Center Grant No 201725NNZ902915 the statutory activities of the Department of Physics and Biophysics Wroclaw University of Environmental and Life Sciences

References [1] Clifford N Journal of the Science of Food and Agriculture 80 (7) 1063-1072 2000 [2] BendokasV Semiene K et al Critical Reviews in Food Science and Nutrition 13 1-14 2019 [3] Khoo HE Alzan A et al Food amp Nutrition Research 61(1) 1361779 2017


228

Effect of immunosuppressive treatment during pregnancy

on the metabolism of minerals in the hard tissues of female rats

and their offspring

Styburski Daniel1 Wojciech Żwierełło

1 Marta Skoacuterka-Majewicz

1 Justyna Kikut

2

Justyna Kałduńska2 Nina Konecka

3 Natalia Komorniak

2 Patrycja Kapczuk

4

Joanna Palma2

1Department of Medical Chemistry Pomeranian Medical University in Szczecin 2Department of Human Nutrition and Metabolomic Pomeranian Medical University

in Szczecin 3Department of Applied Neurocognitivistic Pomeranian Medical University

in Szczecin 4Department of Biochemistry and Medical Chemistry Pomeranian Medical

University in Szczecin

Background

Organ transplantation is a generally accep-

ted treatment method for organ failure

When successful it can reverse many

complications caused by organ dysfunction

but bone metabolism and mineralisation

disorders continue to be problematic In

order to prevent transplant rejection immu-

nosuppressive therapy is a necessity also for

pregnant transplant recipients which means

that the medications may influence foetal

development Calcium phosphorus and

magnesium are some of the essential

minerals affecting normal bone formation

99 of calcium 85 of phosphorus approx

60 of magnesium in the human body is

found in hard tissues Immunosuppressants

may affect on the content of these elements

Therefore in this study we evaluated the

impact of three regimens of immunosup-

pressive therapy used after renal transplan-

tation on the of the essential minerals

(calcium phosphorus and magnesium)

affecting normal bone formation


The study was conducted on 32 female

Wistar rats Eight rats comprising the con-

trol group were given carrier and olive oil

whereas three study groups receiving drug

combinations Each group consisted of eight

female rats

Group 1 (Therapy 1) ndash receiving CsA

(cyclosporine A) MMF (mycophenolate

mofetil) and prednisone

Group 2 (Therapy 2) ndash receiving Tc

(tacrolimus) MMF and prednisone


everolimus and prednisone

A total of 148 pups were born 54 in control

group 36 in group 1 48 in group 2 and 10

in group 3

Femur and tooth material collected during

necropsy was dried at 100degC until dry mass

was obtained Dry tissue was crushed and

100 mg samples were weighed into plastic

vials and labelled After preparation the

samples were subjected to a microwave

decomposition procedure using a micro-

wave digestion system

The samples were analysed using indu-

ctively coupled plasma optical emission

spectrometry (ICP-OES) equipped with

a concentric nebuliser and a cyclonic spray

chamber

Results

The immunosuppressive regimens under

study had no effect on the levels of mag-

nesium and phosphorus but they did

contribute to increased bone calcium levels

of mother rats moreover no changes were

identified in the levels of the studied

229

minerals in the teeth of mother rats The

therapy 1 increases bone magnesium levels

in the offspring while a therapy 3 signifi-

cantly reduces the magnesium level in the

teeth of the offspring


The immunosuppressive regimens did not

affect the levels of magnesium and phos-

phorus in the rat model (mothers) but they

did contribute to an increased bone calcium

level No changes were identified in the

levels of the studied minerals in the teeth of

mother rats which may suggest a protective

effect of the studied regimens on hard

tissues The use of prednisone CsA and

MMF increases bone magnesium levels in

the offspring while a therapy based on

prednisone CsA and everolimus signify-


teeth of the offspring potentially affecting

mineralisation and strength of hard tissues

References [1] Kalantar-Zadeh K Molnar MZ Kovesdy CP et

al (2012) Management of mineral and bone dis-

order after kidney transplantation Current Opinion

in Nephrology and Hypertension 21389-403

httpsdoiorg101097MNH0b013e3283546ee0

[2] Kabat-Koperska J Kolasa-Wołosiuk A

Wojciuk B et al (2016) The influence of

intrauterine exposure to immunosuppressive

treatment on changes in the immune system in

juvenile Wistar rats DDDT Volume 102279-2288

httpsdoiorg102147DDDTS102189

[3] Shah S Verma P (2016) Overview of Pregnancy

in Renal Transplant Patients International Journal

of Nephrology20161-7 httpsdoiorg101155

20164539342

[4] Palacios C (2006) The Role of Nutrients in Bone

Health from A to Z Critical Reviews in Food

Science and Nutrition46621-628 httpsdoiorg

10108010408390500466174

[5] Kovacs CS (2015) Calcium phosphorus and

bone metabolism in the fetus and newborn Early

Human Development 91623-628 httpsdoiorg1

01016jearlhumdev201508007


The expression of virulence- and biofilm-related genes in selected

species of uropathogenic Gram-negative rods

Z Sycz1 M Szydłowicz

1 M Kabaj

2 K Klimas


23

D Wojnicz1

1Department of Biology and Medical Parasitology Wrocław Medical University Wrocław

Poland 2Department and Clinic of Angiology Systemic Hypertension and Diabetology

Wrocław Medical University Wrocław Poland 3Department of Social Medicine Wrocław

Medical University Wrocław Poland

This paper focuses on the overview of

virulence- and biofilm-related genes which

are present in uropathogenic Gram-negative

rods Escherichia coli (UPEC) Entero-

bacter cloacae and Pseudomonas aerugi-

nosa These bacterial species may be

responsible for acute recurrent and chronic

urinary tract infections (UTIs) also

nosocomial UTIs associated with the use of

urinary catheters (CAUTIs) [1245]

Uropathogenic strains harbour many genes

encoding virulence factors enabling the

bacterium to resist and overcome several

defence strategies of the host and support

different steps in uropathogenesis The first

step in the pathogenesis of UTIs is adhesion

(tight and irreversible adherence) of bacteria

to host uroepithelial tissue [2] Virulence

factors responsible for microbial attachment

include fimbriaepili belonging to the

chaperone-usher family (types 1 P S F1C

230

F9 3 UCL F17G and IV) amyloid curli

fibers AfaDr adhesins non-fimbrial adhe-

sins belonging to the group of autotrans-

porter proteins (Ag43 Upa) and other

afimbrial adhesins (proteins lipopoly-

saccharides) [6] The presence of bacterial

cell surface hydrophobicity and T3SS (type

III secretion system including thiol-

activated pore-forming cytotoxins) or T4SS

(type IV secretion system) can be used as a

general indicator of bacterial virulence [4]

Also structures which are primarily asso-

ciated with other functions may contribute

to bacterial adherence promote adhesion

and invasion eg ability to move (swim-

ming motility) through flagella some

toxins α-haemolysin endotoxin cytotoxic

necrotising factors and serine protease

autotransporters of the Enterobacteriaceae

(SPATEs) [2 4 5]

After adhesion uropathogenic Gram-nega-

tive rods usually form biofilms consisting of

exopolysaccharide- or alginate-surrounded

microcolonies which promote long-term

colonisation and persistence in the urinary

tract Pathogens growing in a biofilm mass

are a serious threat to human health because

of their resistance to immune system factors

and antibiotics [2 4-6]

In uropathogenic bacteria genes encoding

virulence and biofilm factors are typically

located on horizontally acquired mobile

genetic elements (MGE) called pathogen-

nicity islands (PAIs) The organized regu-

lation of gene expression is an important

factor in an appropriate invasion coloni-

zation growth andor toxin production

[1 3-5]

Nowadays the objective of researches is to

investigate the correlation of bacterial

ability to virulence factor expression and

biofilm formation with the number of gene

copies and their mRNA expression Uro-

pathogenic bacterial strains are checked for

the presence of virulence-related genes

andor biofilm-formation-associated genes

using the PCR methods Total DNA is

isolated from overnight bacterial culture

using a bacterial genomic DNA purification

kit All PCR and qRT-PCR reactions are

performed using Taq DNA polymerase The

isolates are screened for the presence

of virulence-related genes and genes impor-

tant for biofilm formation The sequence

coding for 16SrRNA is used as a positive

control PCR amplification products are

separated by electrophoresis in agarose gel

Gel images are visualized and analysed

[1 3-5]

The characterization of virulence- and

biofilm-related genes can be useful to

improve our understanding of the patho-

genesis of UTI to develop effective treat-

ment strategies and to minimize the compli-

cations including kidney failure [1 3 6]

References [1] F Firoozeh M Saffari et al Int J Infect Dis

vol 29219-22 2014 doi 101016jijid

2014031393

[2] P Luumlthje A Brauner Adv Microb Physiol vol

65337-72 2014 doi 101016bsampbs

201408006

[3] M Raeispour R Ranjbar Antimicrob Resist

Infect Control vol 7118 2018 doi 101186

s13756-018-0411-4

[4] J Xu F He J Glob Antimicrob Resist vol

17198-200 2019 doi 101016jjgar201904007

[5] M Estaji M Tabasi et al Comp Immunol

Microbiol Infect Dis vol 6523-8 2019

doi 101016jcimid201903014

[6] D Johnson Bacterial Pathogens and Their

Virulence Factors Springer International Publishing

AG 2018 doi 101007978-3-319-67651-7


231

Toxicity of yohimbine ndash a drug used in erectile dysfunction

and body-building supplements

Patrycja Szczepańska1 Małgorzata Szafraniec

1 Dominika Witkowska

1 Ewa Sawicka

2

1Studentsrsquo Science Club of department of Toxicology Faculty of Pharmacy Wroclaw

Medical University Wroclaw Poland 2Department of Toxicology Faculty of Pharmacy


Introduction

Yohimbine is an alkaloid which is naturally

found in barks of the African tree

Pausinystalia johimbe and South American

Aspidosperma quebracho-blanco tree The

structure of molecule resembles a tryp-

tamine The most known application of

yohimbine is the treatment for impotence In

addition products containing yohimbine are

advertised as a body-building and weight-

loss promoting dietary supplements because

of its lipolytic effect

Mechanism of action

The substance is an alpha 2-adrenergic

receptor antagonist The drug-receptor inte-

raction causes an increased release of norad-

renaline and dopamine By blocking the pre-

and postsynaptic alpha-2 adrenoceptors

yohimbine dilates blood vessels in skin and

genitals An average oral dose of 5-15 mg

produces a therapeutic whole blood level

range of 40-400 ngmL This effect is used

in erectile dysfunction treatment

Lipolytic effect

Studies have shown that yohimbine has an

efficacy in fat lipolysis in the abdominal

area Alpha-2 receptors are found predo-

minantly at the site of resistant fatty tissue

They cause that lipolysis is inhibited which

prevents the oxidation of fat cells and

weight loss In addition fat tissue accumu-

lates beta-2 receptors that activate the

lipolysis and cause the oxidation of fat cells

Yohimbine blocks alpha-2 receptors which

results in adrenaline moves towards beta-2

receptors which increase fat burning In this

case fat burning begins To achieve the full

potential of yohimbine it should be used if

a person has low body fat but located

in resistant places

Toxicity

Anxiety

Drowsiness

Disorientation

Tremors

Seizures

Fatal case

An unconscious 23-year-old male body

builder was presented to the emergency

room with seizures and elevated vitals and

was pronounced dead within hours Through

investigation jars of yohimbine and caffeine

powder were among supplements recovered

from the decedents residence along with

arginine L-carnitine beta-alanine and

testosterone The decedent had a medical

history of low testosterone and hypogo-

nadism Notable autopsy findings were

cardiomegaly (525 g) pulmonary edema

and congestion

Conclusion

Dietary supplements contrary to pharma-

ceutical drugs are not tested for effecti-

veness nor safety for example chronic

toxicity (as a result of long term exposure)

It is especially important in case of

yohimbine because it is characterized by

a low therapeutic index meaning there is

a small range of therapeutic doses which do

not entail dangerous and potentially fatal

complications

232

References [1] Colin Anderson Dan Anderson Nicole Harre

Norman Wade Case Study Two Fatal Case

Reports of Acute Yohimbine Intoxication Journal of

Analytical Toxicology Volume 37 Issue 8 October

2013 Pages 611-614

[2] Collins S Surwit RS The beta-adrenergic

receptors and the control of adipose tissue

metabolism and thermogenesis Recent Prog Horm

Res 200156309-28

[3] Johan Ruud amp Jens C Bruumlning Light on leptin

link to lipolysis Nature volume 527 pp 43-

44(2015)


Characteristic of bacteriophage phi80-18 infecting Yersinia

enterocolitica

Bożena Szermer-Olearnik1 Karolina Filik

1 Anna Wroacuteblewska

2 Karolina Kichler

1

Ewa Brzozowska1


Wroclaw Poland 2 University of Wroclaw Faculty of Biotechnology Wroclaw Poland

Background

Yersinia enterocolitica is a gram-negative

bacterium that causes disease called

yersiniosis The infection is manifested as

acute diarrhea mesenteric adenitis terminal

ileitis and pseudoappendicitis [1] Most

pathogenic for humans is O3 serotype

(occurring mainly in Europe and China) and

O8 serotype (occurring mainly in the USA)

The migration of people between continents

is taking place without any obstacles that is

why the occurrence of Y enterocolitica in-

fections in Europe becoming more frequent

The use of bacteriophages is being consi-

dered as an alternative method to control

pathogenic bacteria because they charac-

terized exceptional specificity infecting and

destroying only the host bacterial cells [2]


To measure thermal and pH stability

bacteriophage phi80-18 was incubated in

wide temperature range between 4-80degC

and in the pH range between 2-12 The

phage titer was evaluated by the double-

layer agar method Bacteriophage phi 80-18

was visualized using a JEOL JEM-1200 EX

80 kV TEM Phylogeny of Yersinia phage

phi80-18 was inferred independently using

whole genome data and RNA polymerase

(RNAP) protein sequence as a phylogenetic

marker

Results

During our research we characterize pre-

viously sequenced Yersinia enterocolitica

bacteriophage phi80-18 showing a wide pH tolerance range and stability at relatively

high temperatures Phylogenetic trees recon-

structed using whole genome sequence or

RNAP protein sequence confirmes that

phi80-18 belongs to Autographivirinae

subfamily in Podoviridae TEM analysis of

viral morphology confirms that phi80-18

belongs to Podoviridae family and shows

that the estimated virion length is within

70 nm


Described properties and wide range of

tolerance makes bacteriophage phi80-18 an

excellent tool to fight with pathogenic bac-

teria in the field of biotechnology industry

For example it can be useful when we think

about using phages in food processing

industry where bacteriophage biocontrol

may become natural method that uses lytic

bacteriophages isolated from the envi-

ronment to specifically target pathogenic

bacteria [3]

233

Research financed by the NCN Sonata Bis 7

UMO-201726ENZ100249

References [1] P Raymond E Houard et al Microbiology-

open 8(6)e00751 2019 DOI 101002mbo3751

[2] R Garcia S Latz et al Front Microbiol 10

1187 2019 DOI 103389fmicb201901187

[3] ZD Moye J Woolston et al Viruses 10(4)

205 2018 DOI 103390v10040205


Optimal conditions for efficient formation of PAMAM dendrimers

complexes with 5-fluorouracil

Magdalena Szota1 Jordan Mooney

2 Barbara Jachimska

1

1 Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences

Krakow Polan 2Chemical and Process Engineering University of Strathclyde Glasgow

United Kingdom

Background

In todays growing number of cancer cases

more effective methods for fighting cancer

are being sought One possible method is to

use a drug carrier to increase the effect-

tiveness of the therapy and eliminate side

effects present during pharmacotherapy In

this work poly(amidoamine) (PAMAM)

dendrimers were explored as a potential

carrier for 5-fluorouracyl a drug used mainly

in the treatment of colorectal cancer [1]

Dendrimers are synthetic polymers with

well-defined size and structure Due to their

nano-size shape and controllability dendri-

mers have many applications in biotech-

nology and seem to be an ideal carrier [2 3]

Two generations of positively charged

dendrimers (4th and 6th) have been studied

here looking for the best conditions for the

formation of an effective dendrimer-drug

complex by varying molar ratio ionic

strength and pH


Experiments were conducted on 4th and 6th

generation poly(amidoamine) PAMAM

dendrimers with ethylenediamine-based

core and terminal NH2-groups (Dendritech

Inc Michigan Midland MI USA)

5-Fluorouracil was purchased from Sigma-

Aldrich The solutions were prepared in

deionized water and sodium chloride

(NaCl)

Analytical techniques were used which

allowed the determination of the loading

efficiency (UV-Vis spectroscopy) particle

size and zeta potential (Dynamic Light

Scattering) and the effectiveness of

dendrimer and drug adsorption (Quartz

Crystal Microbalance with Dissipation

monitoring) Additionally the contact angles

were determined for the dendrimer layer

deposited on the gold sensor (KRUSS)

Results

The complex was prepared under various

conditions and the efficiency of binding

ligand to dendrimer after dialysis was

determined by UV-Vis spectroscopy The

analysis showed that the efficiency of

binding ligand to dendrimer is strictly depen-

dent on the conditions of complex for-

mation molar ratio ionic strength and pH

Higher efficiency was observed during the

application of molar excess of the drug in

relation to the dendrimer As far as other

parameters are concerned the study showed

that higher efficiency occurs at lower ionic

strength and higher pH

234

Using the method of Dynamic Light Scat-

tering dendrimer particle sizes were deter-

mined for both generations The analysis of

the particle size of the complex showed the

formation of aggregates during the forma-

tion of the complex

Zeta potential measurement was performed

to characterise the dendrimers and used to

determine the isoelectric point The research

has shown that zeta potential decreases for

higher ionic strength at the same pH but the

isoelectric point remains unchanged

The QCM-D measurement was using to

measure the effectiveness of dendrimer

adsorption and the numbers of drug mole-

cules immobilized in the dendrimer structure

[2] Used QCM-D we were looking for the

best conditions and the optimal molar ratio

for the formation of the complex


The results show that poly(amidoamines)

dendrimers can be active carriers of

5-fluorouracil for uses in biomedical appli-

cations in cancer treatment Optimal condi-

tions and their effects on loading efficiency

for certain parameters have been deter-

mined Future work will be focused on

characterisation and optimisation of condi-

tions for formation of dendrimer and drug

complexes

Acknowledgments

This work was partially supported

by project NCN OPUS 201623B02788


References [1] K Tokarczyk B Jachimska Colloids and

Surfaces A vol 561 357-363 2019 httpsdoiorg

101016jcolsurfa201810080

[2] B Jachimska M Łapczyńska et al Journal

of Physical Chemistry C vol 117 1136-1145 2013

DOI 101021jp307832p

[3] B Jachimska Nanoparticles in Pharma-

cotherapy 251-274 2019 DOI 101016b978-0-

12-816504-100003-x101016b978-0-12-816504-

100003-x


Effect of Salvianolic Acid B on human gingival fibroblast proliferation

Urszula Szwedowicz1 Anna Choromanska

2

1Faculty of Pharmacy Medical Laboratory Diagnostics Wroclaw Medical University 2Department of Molecular and Cellular Biology Faculty of Pharmacy Wroclaw Medical

University

Background

The anti-oxidative and anti-inflammatory

activities of Salvianolic acid B were

presented in many in vitro and in vivo

studies This water-soluble compound was

isolated and purified from the crude extract

of Salvia miltiorrhiza the pharmacopoeial

plant commonly known in east Asia Here

we wanted to take a step further and invest-

tigate whether Salvianolic acid B can cause

an increased cell proliferation as well To

check this possibility human primary

gingival fibroblast cell line was pre-treated

with Salvianolic acid B in six different

grading concentrations Cell viability was

analysed via the MTT assay after three

different times of incubation with com-

pound Additionally the expression of

collagen III protein after Salvianolic acid B

incubation was assessed by immuno-

cytochemical method


Human gingival fibroblasts were isolated

from a patient As the main method we used

MTT assay after 24 48 and 72 hours

incubation to investigate cell proliferation

Salvianolic acid B tested concentrations

were 25 microgml 50 microgml 75 microgml 100

235

microgml 150 microgml and 200 microgml To

observe collagen III expression changes we

performed immunocytochemistry assay

using rabbit polyclonal collagen III antibody

(ab7778)

Results

The MTT assay showed that Salvianolic

acid B has a stimulating impact on human

fibroblast proliferation There is a corre-

lation between compound concentration and

amount of newly formed cells


The results are promising and can lead to

further clinical research on inter alia

wound healing process

References [1] Waldemar Buchwald Elżbieta Hołderna-

Kędzia Aktywność mikrobiologiczna wyciągu

etanolowego z korzeni szałwii czerwono-

korzeniowej (Salvia miltiorrhiza Bunge) uprawianej

w Polsce Borgis - Postępy Fitoterapii 32007

s 133-135

[2] Yong Fan Qianping Luo Mechanism of

salvianolic acid B neuroprotection against

ischemiareperfusion induced cerebral injury Brain

Research Volume 1679 15 January 2018 Pages

125-133


Pneumocystis pneumonia ndash who should be vigilant

M Szydłowicz1 M Kicia

1 Ż Zajączkowska

1 Z Sycz

1

1Department of Biology and Medical Parasitology Wrocław Medical University Mikulicza-

Radeckiego 9 50-345 Wrocław Poland

This paper reviews the risk of infection of

an opportunistic pulmonary fungus

ndash Pneumocystis jirovecii ndash in different

groups of patients Immunocompromised

individuals constitute the main group of risk

of infection and development of symptoms

of Pneumocystis pneumonia (PCP or

pneumocystosis) PCP used to be the most

common AIDS-defining infection in 1980s

however due to the introduction of anti-

retrovirus therapy its incidence in HIV-

infected individuals has significantly

decreased since then Nowadays patients

with immunosuppression caused by other

factors than HIV infection are considered

the main group of risk such as eg trans-

plant recipients receiving anti-rejection

drugs cancer patients undergoing chemo-

therapy patients with inflammatory and

rheumatic diseases as well as preterm

infants In these cases the symptoms of PcP

(unproductive cough low-grade fever

progressive dyspnoea) are usually charac-

terized by rapid onset faster progression

and poorer prognosis as compared to HIV-

patients Moreover even very low intensity

of infection may be sufficient to cause

symptoms of PcP which can even lead to

death Therefore it is extremely important

to develop sensitive and specific diagnostic

methods in order to detect even the low

level of the pathogen in patients samples

and introduce specific therapy prior to the

development of this serious disease

In turn the immune system of healthy

individuals usually eliminates the infection

quickly however Pneumocystis may persist

in lungs in the asymptomatic form Since

the transmission of this pathogen occurs via

the airborne route such carriers serve as

a source of infection in the human popu-

lation posing a risk for immunocompro-

mised individuals Therefore colonization

although asymptomatic is also an important

epidemiological issue Furthermore even

individuals without immunosuppression

236

such as patients with various pulmonary

diseases can be more susceptible to infec-

tion due to specific factors

The present review focuses on different

factors affecting the risk of P jirovecii

infection and PcP development including

type of underlying disease drugs used co-

infections and comorbidities

References [1] M-C Li N-Y Lee et al Pneumocystis jiroveci

pneumonia in immunocompromised patients

delayed diagnosis and poor outcomes in non-HIV-

infected individuals J Microbiol Immunol Infect

vol 47 42-45 2014

[2] F Roblot C Godet et al Analysis of underlying

diseases and prognosis factors associated with

Pneumocystis carinii pneumonia in

immunocompro-mised HIV-negative patients Eur J

Clin Microbiol vol 21 253-531 2002

[3] R M Selik E T Starcher et al Opportunistic

diseases reported in AIDS patients frequencies

associations and trends AIDS vol 1 175-182

1987

[4] M Sokulska M Kicia et al Pneumocystis

jirovecii ndash from a commensal to pathogen clinical

and diagnostic review Parasitol Res vol 11 3577-

3585 2015

[5] M M Ward F Donald Pneumocystis carinii

pneumonia in patients with connective tissue

diseases the role of hospital experience in

diagnosis and mortality Arthritis Rheum vol 42

780-789 1999


Interleukin 13 as an immunomodulator in various disease

B Szymańska1 A Chrząstek

2 A Piwowar

1

1Department of Toxicology Medical University in Wroclaw Poland 2Students Scientific

Society at the Department of Toxicology Medical University in Wroclaw Poland

There is a growing evidence that chronic

inflammation may play a key role in

thepathogenesis and development of various

diseases including cancer IL-13 is a

pleiotropic cytokine with anti-inflammatory

and immunoregulatory activity [1]

Literature data indicates its role in the

pathogenesis of cancer such as breast cancer

ovarian cancer pancreatic cancer colorectal

cancer head and neck cancer bladder

cancer and lymphoma cancers However

the results of some studies indicate a contra-

dictory role of IL-13 in promoting and

fighting the progression of cancer The

involvement of IL-13 in the escape of tumor

cells from host immune surveillance is

important [2 3]

In addition IL-13 is found to be involved in

other disease such as parasitic infections

asthma atopy nephrotic syndrome gastro-

intestinal tract diseases and arthritis [4]

Further research especially on the role of

pro-cancer or anti-cancer IL-13 will con-

stitute an important prognostic andor diag-

nostic aspect The use of IL-13 inhibitors in

targeted immunotherapy is also being

considered [5 6]

The aim of this work was to analyze

literature data on the importance of IL-13 as

an anti-inflammatory immunomodulator in

various disease states and potential

mechanisms of its action

References [1] Wynn TA IL-13 Effector Functions Annu Rev

Immunol 21425-456 2003 DOI101146

annurevimmunol21120601141142

[2] May RD Fung M Strategies targeting the IL-

4IL-13 axes in disease Cytokine 75 89-116 2015

DOI 101016jcyto201505018

[3] Kaskas NM Moore-Medlin T et al Serum

biomarkers in head and neck squamous cell cancer

JAMA Otolaryngol ndash Head NeckSurg140 5-11

2014 DOI101002hed23842

237

[4] Marone G Granata F et al The Intriguing Role

of Interleukin 13 in the Pathophysiology of Asthma

Front Pharmacol2019 httpsdoiorg103389

fphar201901387

[5] Foerster J Molęda A et al Feasibility Analysis

of Interleukin-13 as a Target for a Therapeutic

Vaccine Vaccines 7 20 2019 DOI103390

vaccines7010020

[6] Suzuki A Leland P et al Targeting of IL-4 and

IL-13 receptors for cancer therapy Cytokine 75

79-88 2015 DOIorg101016jcyto201505026


Analysis of interleukin 13 and angiogenin in patients with bladder cancer

Szymańska B1 Jurkowska K

1 Sawicka E

1 Matuszewski M

2 Dembowski J

2

Piwowar A1

1Faculty of Pharmacy Department of Toxicology Wroclaw Medical University Poland 2Department of Urology and Urological Oncology Wroclaw University Hospital Poland

Background

Participation interleukin 13 (IL-13) in the

process of carcinogenesis was well studied

but we have only few reports on its invol-

vement in bladder cancer (BC) Angio-

genesis (formation of new blood vessels)

plays a key role in the process of tumour

growth and metastasis It enables the deli-

very of nutrients and oxygen to cancer cells

The aim of the study was to investigate the

role of IL-13 as an anti-inflammatory

immunomodulator and angiogenin (ANG)

as a stimulator of the angiogenesis process

in patients with BC


The concentration of IL-13 and ANG in the

plasma of BC patients and healthy controls

were measured by enzyme-linked immuno-

sorbent assay These parameters were

examined in the whole group of BC patients

and in subgroups depending on clinical

stage non-muscle invasive bladder cancer

(NMIBC) muscle invasive bladder cancer

(MIBC) histopathologic malignancy low

grade (LG) high grade (HG) and in primary

and recurrent BC To assess the IL-13 and

ANG diagnostic value ROC curves were

plotted cut-off points as well as sensitivity

and specificity were calculated The

research was approved by the Bioethics

Committee No (KB-2922-16)

Results

In patients with bladder cancer were found

significantly higher mean plasma concen-

tration of IL-13 (plt0001) and ANG

(plt0001) compared to the control group

Higher mean IL-13 plasma concentrations

corresponded to lower disease stages

(NMIBC LG) In contrast mean ANG levels

were higher in advanced stages (MIBC

HG) of BC The mean concentration IL-13

and ANG were similar in primary cancer

and recurrence BC


Higher IL-13 expression in bladder cancer

tissues has been demonstrated by

immunohistochemical studies Urinary

levels of IL-13 have also been shown to be

useful as a marker in bladder cancer Higher

serum levels of ANG have been demon-

strated in many types of cancers

In the above study the potential of the

tested indicators as diagnostic parameters in

bladder cancer has been demonstrated

References [1] Foerster J Molęda A et al Feasibility Analysis


Vaccine Vaccines vol 7 20 2019

doi 103390vaccines7010020

[2] Wynn TA IL-13 Effector Functions Annu

Rev Immunol vol 21425-456 2003 doi101146


238

[3] Margel D Pesvner-Fischer M et al Stress

proteins and cytokines are urinary biomarkers for

diagnosis and staging of bladder cancer Eur Urol

vol 59 113-119 2019 doi 101016jeururo

201010008

[4] MalekZadeh K Nikbakht M et al

Overexpression of IL-13 in patients with bladder

cancer Cancer Invest vol 28 201-207 2010 doi

10310907357900903181977

[5] Peres R Furuya H et al Angiogenin contributes

to bladder cancer tumorigenesis by DNMT3b-

mediated MMP2 activation Oncotarget vol 7

43109-43123 2016 doi 1018632oncotarget

10097

[6] Yu D Cai Y et al The Potential of Angiogenin

as a Serum Biomarker for Diseases Systematic

Review and Meta-Analysis Disease markers vol

2018 1984718 doi10115520181984718


Theoretical studies on the structure of fagopyrin

Sebastian Szymański1 Irena Majerz

2


Background

Compounds with a double anthrone moiety

find an use in the treatment of cancer

depression as a natural laxative and against

constipation One of the double anthrone

is fagopyrin a natural ingredient of Fagopy-

rum esculentum Fagopyrin occurs as many

conformers (Fig 1) Physical and thera-

peutic properties of fagopyrin result from its

molecular structure that is not determined

Theoretical methods were used to determine

the geometrical and electronic structure of

fagopyrin conformers


Structures of fagopyrin were optimized

using Gaussian 16 [1] software BLYP6-

311++G(dp)-D3 method was applied to

optimization in the gas phase QTAIM

analysis was performed using AIMALL [2]

program Non-covalent interactions were

described by NCI [3] software

Results

Figure 1 presents the molecular structure of

fagopyrin and the substituents present in the

plant material For fagopyrin A - F and

different orientation of hydroxyl group the

structure has been optimized and many

conformers characterized by different ener-

gy have been found Theoretical methods

allowed to determine the intramolecular

interactions in the fagopyrin derivatives

QTAIM and NCI analysis indicates the

strong OHO hydrogen bonding in the

anthrone moiety The presence of substi-

tuents containing nitrogen atom allows

formation of OHN hydrogen bond linking

hydroxyl group in the anthrone moiety and

the nitrogen atom in the cyclic substituent


The system of the strong O-HhellipOhellipH-O

hydrogen bonds in the anthrone molecule

can be changed under formation of O-HhellipN

hydrogen bond to the nitrogen substituent

Specific properties and numerous appli-

cation of a double anthrone compounds

result from their molecular and electronic

structure Molecular structure of fagopyrin

is unexplored thus the use of theoretical

methods allows to approximate its structure

that determines physicochemical properties

References [1] Gaussian 16 Revision C01 Gaussian Inc

Wallingford CT 2016

[2] AIMAll (Version 191012) Todd A Keith TK

Gristmill Software Overland Park KS USA 2019

(aimtkgristmillcom)

[3] Contreras-Garcia J et al J Chem Theory

Comput (2011) doi101021ct100641a

[4] Benković E T Žigon D Friedrich M

Plavec J amp Kreft S FoodChem (2014)

doi101016jfoodchem201307118

239

Figure 1 Possible conformers of fagopyrin [4]


Theoretical study of monoanthrones

Małgorzata Szymańska1 Irena Majerz

2

1Faculty of Pharmacy Medical University in Wrocław 2Faculty of Pharmacy Medical

University in Wrocław

Background

Monoanthrones are tricyclic compounds of

plant origin Due to their wide biological

activity they are used in both herbal

medicine and medicine An interesting

feature of monoanthrones is their non-planar

structure The therapeutic properties of

monoanthrones result from the geometrical

and electronic structure For better under-

standing of the mechanism of drug action

the effect of substitution in the aliphatic ring

and in the side ring on the geometry has

been studied


Optimization of the geometry of several

series of compounds with different substi-

tuents in the rings was carried out The

investigated molecules were optimized

using a Gaussian 16 package at DFT

B3LYP6-311++G level which including

Grimme dispersion Calculations of electron

density were performed using the AIMALL

program Aromaticity expressed by the

HOMA (Harmonic Oscillator Model of

Aromaticity) parameter for anthrone aro-

matic and aliphatic rings were determined

Results

The change of the angle between the

anthrone aromatic rings is associated with

the change in electron density The central

aliphatic ring takes on a partly aromatic

character after substitution of the side ring

with an electron donating substituent while

substitution of the side ring with an electron

withdrawing groups causes losing partly

aromatic character

240

Discussion and conclusions Aromaticity of the anthrone rings is affected by the electron donating and electron withdrawing properties and size of the substituent linked to the aromatic side ring as well as to the central aliphatic ring

Substituents in the aromatic ring affect the geometry and electronic structure of the central ring

Substitution in the central ring have the greatest impact on the structure of the entire molecule

References [1] Habtemariam S Antioxidant activity of Knipholone anthrone Food Chem (2007) 102(4)1042-1047 doi101016jfoodchem2006 06040 [2] Krygowski T M Crystallographic Studies of Inter- and Intramolecular Interactions Reflected in Aromatic Character of π-Electron Systems J Chem Inf Comput Sci (1993) 33 1 70-78 doi101021ci00011a011 [3] MJ Frisch et al Gaussian Inc 16 Revision A03 Wallingford CT (2016)


The spectroscopic studies of the interaction between human

apo-transferrin and copper(II) complexes based on 29-dimethyl-110-

phenanthroline and 135-triaza-7-phosphaadamantane-7-oxide

Urszula Śliwińska-Hill Martyna Matoszka

Faculty of Pharmacy Wrocław Medical University Borowska 211 Wrocław Poland

Background Spectroscopic studies involving transition metal complexes and blood proteins such as human serum transferrinalbumin are essen-tial for the understanding of the biological activity of the drugs in terms of nature and strength of the interactions [1 2] Such interactions may affect the concentration and deactivation of the drug and thereby influence its availability and toxicity during chemotherapy [3] For example when cisplatin is introduced into the body intra-venously 50minus61 of platinum is bound to HSA [45] and the binding is essentially irreversible with less than 5 loss of bound-platinum after extensive dialysis It is very important from the pharmacological point of view because only unbounded with protein part of the drug is biologically active In this context the subject of our research is the analysis of the interactions between biolo-gically active copper (II) complexes and human serum apo-transferrin

Material and Methods All compounds (1 2 3) were synthesized by prof Piotr Smoleński and co-workers from Faculty of Chemistry University of Wrocław

Apo-transferrin (ge 98 Sigma-Aldrich) was used without prior purification The final solutions were prepared in PBS (pH 740) in the molar ratios (protein)(drug) = 10ndash116 with the protein concentration equal 2 microM Samples were incubated at 300 and 310 K for 5 min

Emission fluorescence spectra were recor-ded on Jasco 8200 spectrofluorimeter in the range of 300ndash500 nm using 10 cm quartz cells and the λex = 280 All fluorescence intensities were corrected for the inner filter and dilution effects and the corrected values were used to determine the quenching mechanism and binding data Moreover the copper complexes showed a fluorescence signal in the measured range Therefore all spectra are shown as different spectra of (apo- Tf-copper complex)-(copper complex)

241

Circular dichroism measurements were carried out on a Jasco J-715 spectropolarimeter in the range of 190-250 nm using 01-cm cuvettes

Results Under physiological conditions titration of the protein with small amounts of the comp-lexes caused distinct decrease in fluores-cence intensity of the protein and the maximum band position was red shifted ca 5 nm It indicates that the apo-Tf confor-mation was changed and the proteinrsquos chro-mophores were moved to a more polar environment Moreover based on the Stern ndash Volmer equation it was shown that protein fluorescence quenching by 12 complexes was initiated by both static and dynamic processes and it was static process in the case of 3

Under tested conditions only one binding site (n) in protein for all copper complexes exists The association constants (Ka) decre-ased with the temperature increases for 1 and 2 suggesting formation of the unstable complexes The binding constant of apo-Tf ndash 3 system increased with temperature increase indicating the formation of the stable adduct and endothermic process

The interaction of 1 and 2 with apo-Tf had an insignificant effect on its secondary structure and upon binding the complexes

α-helix content decreased about 1-2 respectively when the molar ratio apo-Tf12 was 120 In contrast to 1 and 2 binding 3 complex to apo-transferrin caused extensive changes in conformation of the protein reducing α-helix content about 12 at the molar ratio apo-Tf3 equal 120

Discussion and conclusions All tested compounds interact with human apo-transferrin causing a conformational changes of the protein Complex 3 showed the most extensive interaction with the loss of helical stability of the protein The positive values of ∆S0 and negative ∆H0 for apo-Tf ndash12 systems indicated electrostatic interactions and both positive parameters for 3 revealed hydrophobic and ionic inter-actions Moreover all reactions between copper complexes and human apo-trans-ferrin were spontaneous processes

References [1] Sarkar B Prog Food Nutr Sci vol 11 363-400 1987 [2] Bal W Christodoulou J et al J Inorg Biochemvol 70 33-39 1998 [3] Espoacutesito BP Najjar R Coord Chem Rev vol 232 137-149 2002 [4] Hu W Luo Q etal Chem Commun vol 47 6006-6008 2011 [5] Zheng Y-R Suntharalingam K etal J Am Chem Soc vol 136 8790-8798 2014


Persistent organic pollutants impact on the incidence of type 2 diabetes

Agnieszka Święcicka-Klama12


1 Zuzanna Sycz

3


Persistent organic pollutants (POPs) are organic chemicals that are toxic and highly resistant to any kind of environmental degra-dation Most of them are currently or were in the past produced as pesticides insecti-cides herbicides solvents flame retardants

and as a chemical intermediates Also gene-rated unintentionally during combustion thermal and industrial processes Although a lot of these compounds have been banned or placed under international control POPs are still common in the environment and

242

accumulate in soil sediments and the food chain People are still exposed to the toxins mostly through their diet particularly fatty animal food such as meat fish and dairy products

The number of studies have reported poten-tial associations between this exposure to POPs and various harmful health effects including cancers increased birth defects neuroendocrine disruption reproductive problems and metabolic disorders like obe-sity and diabetes type 2 [156] The aim of this paper was to assess the impact of POP exposure on the incidence of type 2 diabe-tes based on a review of the current literature

Numerous epidemiological and cross-sec-tional studies indicate a correlation between POP serum level and the development of type 2 diabetes [1 2 4-6] It points to certain polychlorinated biphenyls (PCBs) and several organochlorine pesticides (OCPs) in particular dichlorodiphenyltri-chloroethane (DDT) and dichloro-diphe-nyldichloroethylene (DDE) as potentially adversely affecting substances [1 4] Due to their high lipophilicity and persistence in the body the POP serum level increase with age However the POP serum levels varies a lot between studies and the results are inconsistent [1 4-6] Differences in study population POP exposure distributions varying methodology presentation and analysis of the results could partly explain these discrepancies [1 3-5] Thus the lowest published toxic dose (TDLO) of indi-vidual POP compounds remains uncertain

The pathophysiology mechanism under-lying between POP exposure and develop-ment of diabetes also seems to be complex Some researchers suggest that POPs are associated with insulin resistance impaired glucose uptake metabolic syndrome and abdominal obesity [2 3] Other studies indicate on beta cell function impairment [1 6] Unfortunately the long period between exposure and development of health prob-

lems the influence of a range of other environmental factors and possibly additive effects of the POP compound mixture hamper investigation [35]

In conclusion numerous studies reported a strong correlation between serum concen-tration of POPs (especially organochlorine compounds) and diabetes However there is a strong need for further animal and in vitro research to clarify the impact of POPs on the incidence of type 2 diabetes In the face of the rapid increase in diabetes prevalence and highly developed industry understand-ding the role of environmental chemicals like POPs in the development of diabetes is an emerging issue

References [1] De Tata V Association of dioxin and other persistent organic pollutants (POPs) with diabetes Epidemiological evidence and new mechanisms of beta cell dysfunction Int J Mol Sci 2014 15(5) 7787-7811 doi103390ijms15057787 [2] Grice BA Nelson RG Williams DE et al Associations between persistent organic pollutants type 2 diabetes diabetic nephropathy and mortality Occup Environ Med 201774(7)521-527 doi101136oemed-2016-103948 [3] Kim YA Park JB Woo MS Lee SY Kim HY Yoo YH Persistent organic pollutant-mediated insulin resistance Int J Environ Res Public Health 201916(3)1-14 doi103390ijerph16030448 [4] Taylor KW Novak RF Anderson HA et al Evaluation of the association between persistent organic pollutants (POPs) and diabetes in epidemiological studies A national toxicology program workshop review Environ Health Perspect 2013121(7)774-783 doi101289 ehp1205502 [5] Tornevi A Sommar J Rantakokko P et al Chlorinated persistent organic pollutants and type 2 diabetes ndash A population-based study with pre- and post-diagnostic plasma samples Environ Res 201917435-45 doi101016jenvres201904017 [6] Wolf K Bongaerts BWC Schneider A et al Persistent organic pollutants and the incidence of type 2 diabetes in the CARLA and KORA cohort studies Environ Int 2019129221-228 doi10 1016jenvint201905030


243

Edible Insects as Bread Making Ingredients

Nathan Tancula1 Joanna Harasym

12 Karol Banaś

1 Agnieszka Orkusz

12 Remigiusz

Olędzki12




The use of edible insects as a protein source

in food stretches back for millennia how-

ever the practice has yet to win over

European and Western Consumers industry

regulators and sanitary regulators Further-

more the economic viability and environ-

mental impacts of the harvest of insects has

yet to be proven Within the laboratory the

use of edible insect flour in both conven-

tional wheat-based flour bread and gluten

free bread has shown favourable results in

overall protein fat and structural effects on

the nutritional content of these types of

bread

The overall water absorption capacity was

shown to be reduced when using insect

flours ranging from 45-57 along with fat

contents from 27-36 [1] However the

bread making process could be carried out

with all composite flours showing similar

texture and volume parameters when com-

pared to conventional wheat bread yet with

higher protein and fibre concentrations

attributed to insect flours

Results confirm that the enrichment with

cricket powder can lead to the production of

gluten free bread with acceptable technolo-

gical properties and high protein content [2]

Tests have proven that the porous structure

which is the signature of conventional

wheat bread can be reproduced using

a gluten-free batter containing insect-deri-

ved flour The porous structure can be attri-

buted to the insect flourrsquos protein and lipid

contribution to the mixture

When sensory and overall cultural accep-

tance is taken into consideration a bread

with 10 cricket flour showed a global

liking by untrained panellists [3]

References [1] Gonzaacutelez C M Garzoacuten R amp Rosell C M

(2019) Innovative Food Science and Emerging

Technologies 51(January 2018) 205-210 101016

jifset201803021

[2] da Rosa Machado C amp Thys R C S (2019)

Innovative Food Science and Emerging Techno-

logies 56(May 2019) 101016jifset2019102180

[3] Osimani A Milanović V et al Innovative

Food Science and Emerging Technologies

48(June) 150-163 101016jifset201806007


Functional properties of blends of rice and house cricket

(Acheta domesticus) flours


12 Karol Banaś


12

Agnieszka Orkusz12




244

Background

The demand for gluten-free products has

been rising as the total number of consu-

mers avoiding wheatgluten-containing pro-

ducts increases exponentially [1] Mean-

while the main problem of gluten-free

breads is the fact that they are overloaded

with structuring agents the main purpose

of which is to create the spongyporous

structure found in typical - wheat breads

from ingredients which do not contain the

gluten-protein [1] The predominant ingre-

dient in a gluten-free formula is starch

while native flours are few The more

frequently used flour is rice flour which

is rich in starch but has an insufficient

protein level Edible insects are a good

source of protein and can also be used

as nutritional enrichment in gluten-free

formulations

The main objective of this study was to

investigate the hydration properties of rice

flour powdered house cricket blends in

order to assess its suitability as gluten-free

baking ingredient


Hydration properties like water holding

capacity (WHC) oil absorption capacity

(OAC) water solubility index (WSI) along

with the water absorption index (WAI) in

gluten-free flour mixtures composed of

varied blends of rice flour and house cricket

flour were studied Both flours where

purchased commercially on the local market

(Wrocław Poland) and six mixtures were

prepared to contain 5 10 15 20

25 and 30 of cricket flour along with

two controls of 100 rice and 100 cricket

flours

Results

The results revealed a very interesting

behaviour of cricket flour The water hol-

ding capacity in cricket flour sample alone

was significantly higher than in rice flour

one (248 gg db vs 302 gg db) but its

impact in the studied blends was not signi-

ficantly different Also the water soluble

index of cricket flour was the highest among

the studied samples while the water absorp-

tion index was the lowest showing different

water maintaining behaviour after heating

Furthermore the water solubility index of

rice flour was not impacted by cricket flour

addition Furthermore OAC was shown to

be higher using the 100 cricket flour

control as compared to the 100 rice flour

control Thus a gradual increase of oil

absorption capacity was observed starting at

an average of 1478057316 gg db at a 5

Cricket blend to an average of 1507978905

gg db at a 30 Cricket blend


Powdered house cricket can be applied as

baking ingredient because of its high nutria-

tional value As baking ingredient espe-

cially in gluten-free bread-making the specific

water hydration properties make powdered

house cricket a demanding raw material

Hydrophilic and hydrophobic interactions

among bread ingredients result in batter

structure formation and further in crumb

porosity of resulting bread High protein and

fat content of Acheta domesticus powder

revealed more hydrophobic behaviour while

chitin acting like fibre absorbs water

The obtained results open very promising

field for successful application of house

cricket powder as valuable ingredient in

gluten-free breadmaking and promising

perspective as contributor of important

nutrients

References [1] A Osimani V Milanović et al Innovative Food

Science amp Emerging Technologies Volume 48

August 2018 Pages 150-163 101016jifset

201806007


245

Connection between laterality and primary reflexes balance sensory

profile in group of children from 4 to 7 years old

Marlena Telenga1 Anna Pecuch

1 Ewelina Wolańska

1 Ewa Gieysztor

1

1Faculty of Health Sciences Wroclaw Medical University

Background

Laterality is functional domination of one

cerebral hemisphere Any dysfunction of

cerebral hemispheres work could involve

problems with balance concentration and

psychomotor activity The aim of the study

is to verify is it any connection between

type of laterality and primary reflex inte-

gration balance and sensorimotor profile


Fourty nine healthy children aged 4-7 years

were examined In this group was define the

type of laterality of hand leg eye and ear

In each category children were divided into

two groups ndash left and right laterality Their

primitive reflexes were investigated by

S Goddardrsquos test During the research time

of stand on one leg (to check the balance)

was measured and sensorimotor profile was

also defined

Results

Group of children with dominance of left

leg have more problems with control their

preferred leg and also more difficulties with

keeping stable position at one leg stand test

That makes they stand shorter at dominant

(left) leg than children with dominance of

right leg In group of left-leg children is also

more active forward tonic labyrinthine

reflex (TLR) Children with dominance of

left eye have more symptoms of dyspraxia

than group with right-eye children In this

group is also high activity of TLR in exten-

sion In group with dominance of left hand

most of children have auditory hyper-

activity It is also very common in group of

children with crossed laterality There are

no important differences between examined

group of children with left and right domi-

nance of ear


Research shows correlation between late-

rality and activity of primitive reflexes

balance and sensorimotor profile There is

worse level of maturity of nervous system

in examined group of children with left side

domination and crossed laterality

References [1] M Bogdanowicz Leworęczność u dzieci WSiP

1989

[2] E Z Gieysztor L Sadowska et al Nursing and

Public Health vol 26 5-11 2017 1017219

pzp75487

[3] S Goddard-Blythe Neuromotor Immaturity in

Children and Adults The INPP Screening test for

clinicians and health practitioners John Wiley

amp Sons 2015

[4] S Goddard-Blythe Attention balance and

coordination The ABC of Learning Success

John Wiley amp Sons 2009

[5] C Grzywniak Szkoła Specjalna vol 2 98-112

2010

[6] A Huurnink D P Fransz et al Journal of

Biomechanics vol 47 308-312 2014 101016

jjbiomech201310002

[7] A Paczkowska J Szmalec et al Hygeia Public

Health vol 49 531-535 2014

[8] B Prajsner Neuropediatria ed M Kaciński

PZWL 2007

[9] L Sadowska M Dziewulski Neurofizjolo-

giczne podstawy diagnostyki i terapii dzieci

z zaburzeniami rozwojowymi UMK 2012

[10] T Wolańczyk J Komender Zaburzenia

emocjonalne i behawioralne u dzieci PZWL 2005

[11] O Jr Vieira D B Coelho et al Experimental

Brain Research vol 232 269-276 2014 101007

s00221-014-4018-6


246

Smart behaviour of materials used for controlled delivery of bioactive

agents

Beata Tokarek1 Urszula Bazylińska

1

1Department of Physical and Quantum Chemistry Wroclaw University of Science


Numerous limitations connected with tradi-

tional chemotherapy leads to the develop-

ment of the novel concept of Smart Drug

Delivery Systems (SDDSs) It allows to

overcome barriers of conventional treat-

ments such as uncontrollable release of

drugs nonspecific distribution rapid clea-

rance or low bioavailability Moreover

smart nanocarriers make possible to deliver

the bioactive agents to target sites and what

is crucial the drug release rate can be con-

trolled over time [1 2]

Nanoscopic drug delivery systems are

colloidal particles of size less than 500 nm

which possess a high surface area to the

volume ratio They are characterised by

improved effectiveness through usage the

pathological physiology of the tumor tissue

environment ie angiogenesis hypervascu-

larization of vessels feeding the tumor or

reduced pH Furthermore there is better

nanocarrier accumulation at tumor tissues

due to the enhanced permeability and

retention (EPR) effect [1 2]

Currently many studies in nanomedicine

focus on controlled drug release systems

that are sensitive to different types of triggers

what define them as smart or stimuli-res-

ponsive materials They possess the ability

to respond endogenous or exogenous stimuli

including pH temperature enzyme concen-

tration magnetic field light ultrasound

ionic strength or glucose These kinds of ma-

terials are utilized to control the kinetics of

drug release at the specific place and at the

given time [3]

Temperature is one of the most carefully

studied triggers used in drug delivery in

cancer therapies Many polymers are comp-

letely soluble below a certain temperature

referred to as Lower Critical Solution Tem-

perature (LCST) whereas above this point

polymer changes its structure and preci-

pitate from the solution This kind of phase

change can be used for controlled destabi-

lization of polymeric structure and effective

drug delivery [4]

In this research different kinds of smart

behaviour of materials are presented The

kinetic control of drug release possesses

many benefits for patient health including

delivery drug to the specific place with the

reduction of dosage frequencies (the drug

concentration in target sites is constant for

longer time) Due to the number of advan-

tages smart nanocarriers are currently very

promising and intensively studied topic

Acknowledgements

The support of statutory activity subsidy

from the Polish Ministry of Science and

Higher Education for the Faculty of Che-

mistry of Wroclaw University of Science

and Technology is gratefully acknowledged

References [1] D Lombardo MA Kiselev et al Journal of

Nanomaterials vol 2019 1-26 2019 DOI

10115520193702518

[2] S Hossen M K Hossain et al Journal of

Advanced Research vol 15 1-18 2019 DOI

101016jjare201806005

[3] D Liu F Yang et al Theranostics vol 6

1306-1323 2016 DOI 107150thno14858

[4] M Ward T Georgiou Polymers vol 3 1215-

1242 2011 DOI 103390polym3031215


247

The expression of lsquomajor housekeepingrsquo genes decreases in stress

respons ndash is phosphorylation involved

Aleksandra Tomczak1 Marta Pałka


1 Ryszard Rzepecki

1


This work was funded by the Opus 11 grant from the National Science Centre Poland no O-201621BNZ400541

Background The process of transcription is one of the key adaptive mechanisms and needs to be strictly controlled in response to environ-mental factors and stimuli (as heat shock) Recently interest in this topic has been growing among scientists because it is still not known much about the mechanisms controlling it

Heat shock is an invaluable model for stu-dying mechanisms regulating gene expres-sion and is well known and easy to control [1] Many papers report that during stress transcription is shut down globally while only a few loci are highly activated [2] These active loci are connected with heat shock proteins (Hsp) family which functions as intra-cellular chaperones

Lamins are evolutionarily conserved proteins classified as type V intermediate filaments which are involved in the regulation of gene expression chromatin organization DNA replication and repair signaling develop-mental regulation and nuclear positioning [3] In order to play such a variety of func-tions lamins interact with many different nuclear proteins which are directly or indirectly responsible for a particular func-tion Lamins (the main component of the nuclear envelope) together with associated proteins built a complicated platform for the regulation of nuclear processes It has been proved that the chromatin regions located near the nuclear envelope consist mainly of heterochromatin ndash transcriptionally inactive regions

Our research suggest that lamins and asso-ciatedinteracting proteins are significantly

connected with transcription regulation In this work we focus on changes in gene expression profile in stress response Our results also suggest that the phosphorylation status of HSF and lamins changes Does the phosphorylation cause the transcription shut down or is it the result of it

Material and Methods Cell culture and heat shock treatment

All experiments were performed on D melanogaster embryonic cell line ndash Kc Cells were maintained in suspension culture (in Schneider`s Drosophila Medium from Gibco with 10 FBS and 1 antibiotic-antimycotic) at 23degC as normal conditions To induce the heat shock cells were incu-bated at 37degC for 1 h before further experiments

Real-time quantitative PCR and data analysis

Cells were lysed on plates and total RNA was extracted then the cDNA synthesis was performed RNA extractions and cDNA synthesis from all samples were performed for three biological replicates

RT-qPCR was performed using QuantStudiotrade 5 thermocycler and data were calculated by connected Applied Biosystemstrade qPCR analysis module Western blotImmunofluorescence and analysis

Standard western blotimmunofluorescence procedure was performed and data were analyzed using Image LabImageJ software

Results We have developed a protocol that allows us to study the stress response in cells

248

We have found that in response to stress there is a decrease in the expression of transcripts encoding key proteins for cell functioning such as actin tubulin lamins and topoisomerase II In contrast the protein level remains stable

Our data show that ribosomal RNA and transcripts for ribosomal proteins are the most stable and perform well as the refe-rence for studying stress response

We have shown that under stress the phos-phorylation of HSF and lamin Dm occur We also have shown one of a stress-depen-dent phosphorylation site in lamin Dm ndash Ser25

Discussion and conclusions Epigenetics is a field of the future So far many chromatin remodelers with histones and RNA polymerase II in front have been identified as those in which post-trans-lational modifications either activate or lead to gene repression Global transcription shut down is clearly visible in heat shock In this study we show that under stress transcripts level for genes encoding for the key proteins

of the cell decreases eg lamins topoiso-merase II tubulin and actin These trans-cripts are often used as stable references Here we prove that ribosomal RNA-related controls work better

In contrast to data from gene expression protein levels remain stable in stress How-ever the phosphorylation status of proteins is different

In our project we show that during heat shock specific phosphorylation of lamin occurs on Ser25 which results in a change in its solubility and potentially leads to stronger binding of chromatin in stress These data may indicate that lamins play a key role in turning down gene transcription

References [1] J Tower Exp Gerontol 46(5) 355-362 2011 DOI 101016jexger201009002 [2] JF Cardiello JA Goodrich et al Mol Cell Biol 2838(18) 2018 DOI 101128MCB00181-18 [3] M Pałka A Tomczak et al Cell Mol Biol Lett 23 32 2018 DOI 101186s11658-018-0093-1


Colorimetric detection of amino acid sarcosine in urine influence

of zinc (II) ions

Zuzana Toacutethovaacute1 Michaela Všetičkovaacute



1

Branislav Ruttkay-Nedecky2 Warawan Eiamphungporn

3 Josef Růžička

1 Reneacute

Kizek12

1Department of Research and Development Prevention Medicals sro Tovaacuterniacute 342 742 13 Studeacutenka-Butovice Czech Republic 2Department of Human Pharmacology and Toxicology VFU Brno Palackeacuteho třiacuteda 19461 612 42 Brno Czech Republic 3Department of Clinical Microbiology and Applied Technology Faculty of Medical Technology Mahidol University 999 Phutthamonthon 4 Road Salaya Nakhon Pathom 73170 Thailand

Backround The most common cancer in men is prostate carcinoma According to WHO statistics over 11 million new cases are diagnosed worldwide every year Suitable tumor mar-kers are sought for early diagnostics One of the biochemical candidates is the amino

acid sarcosine (SAR) There is an increase in sarcosine levels in the urine In several published studies sarcosine levels ranged from 1 to 25 microM The aim of this work was to optimize the detection of sarcosine in artificial urine

249

Material and methods All chemicals used for this analysis were purchased from Merck (Germany) Used artificial urines were (15 types) AU-N AU-Pro-1 AU-Pro-2 AU-keto-2 AU-pH-1 AU-pH-2 AU-Hemog-1 AU- 1-Brown AU-2-Opalko AU-4-Grases AU-6-Christmas AU-3-Bact AU-5-Mayrovitz AU-7-Chutipongtande The measurement was performed on a pre-washed polystyrene plate (HydroFlex) using a photometer (TECAN Switzerland) The total sample volume was 250 microl and the substrate solu-tion volume was 750 microl The substrate solution contained 4-AAP (4-aminoanti-pyrine) sarcosine oxidase (SOX) horse-radish peroxidase in 02 M phosphate buffer (pH 8) Electrochemical analysis of zinc Zinc chloride was dissolved in phosphate buffer pH 8 The zinc concentration was determined in acetate buffer pH 5 Three-electrode setup (3M Ag AgCl KCl refe-rence electrode glass-carbon auxiliary electrode HMDE working electrode) was used Then the zinc (II) ion solution was added to the substrate solution and mea-sured with the urine sample (25degC) All reactions were documented in the form of photographs The color reaction was evalu-ated and statistically processed in the Labo-ratory Information System Qinslab (Preven-tion medicals Czech Republic)

Results and discussion The Trinder reaction is based on the use of a suitable dye (such as 4-aminoantipyrine) in the presence of hydrogen peroxide and peroxidase Sarcosine oxidase is a flavor-enzyme that is involved in the oxidative demethylation of sarcosine and its reaction with water and oxygen reduces sarcosine to

glycine formaldehyde and hydrogen peroxide

The Trinder reaction produces a quinone imine dye which is photometrically evalu-ated The test without zinc ions was perfor-med with sarcosine additions (216 166 125 625 312 156 and 0 microM) in each of the modified urines (n = 15) Sensitivity in all visually evaluated urines (15 types) ranged from 078 to 1 specificity always corresponded to 1 AUC (area under curve) ranging from 089 to 1 LOD values ranged from 2 to 73 microM in all urine types LOQ values ranged from 9 to 195 microM Subsequently zinc ions (15 mM) were added to the reaction Reaction conditions and sarcosine concentration remained the same Resulting values of reaction with zinc ions (visual evaluation) always correspond-ded to sensitivity 1 specificity 1 AUC also around 1 LOD values ranged from 4 to 53 microM in all urine types LOQ values ranged from 13 to 174 microM In both reactions (with and without ions) the reaction rate was measured The Michaelis Menten constant (according to the Eadie-Scatchard and Lineweaver-Burk method) were twice as high (453) for the zinc ion reaction than for the zinc ion-free reaction (252)

Conclusion Zinc ions in these urines have been shown to support the reaction by up to 100 com-pared to the zinc-free group

Acknowledgements The work was realized with the support of project SarkoTest 1652015 and The Euro-pean Technology Platform for Nanomedicine

References [1] Chutipongtanate S et al Anal Biochem 2010 402(1) 110-112


250

Glymphatic system in pathophysiology of the central nervous system

Michał Tyliszczak1 Marek Kotas

1

1Faculty of Medicine Wroclaw Medical University Wroclaw Poland

Background Glymphatic system plays a vital part in maintaining homeostasis in the central nervous system It not only delivers vital substrates to the brain parenchyma but also clears the parenchyma from unnecessary metabolites This paper focuses on the role of glymphatic system in pathophysiology of the central nervous system and the potential usage of the glympathic system in clinical management

Material and Methods Authors used Google Scholar and Pubmed databases to gather the most recent and reliable sources Following key-phrases were used glymphatic system pathophy-siology central nervous system injury Illustrations were prepared by the authors using computer programs Gimp Inkscape LibreOffice Impress

Results Authors have selected 6 number of articles which best describe recent findings and are most relevant to the possibility of clinical application

Discussion and conclusions As most studies on the glymphatic system are based only on animal models the value of discussed findings is limited especially in regard to potential clinical applications

However the research carried out so far allows us to see the potential for a better understanding of the mechanisms of the glymphatic system

References [1] Jessen N A Munk A et al (2015) The Glymphatic System A Beginnerrsquos Guide Neurochemical Research 40(12) 2583-2599 httpsdoiorg101007s11064-015-1581-6 [2] Piantino J Lim M M et al (2019) Linking Traumatic Brain Injury Sleep Disruption and Post-Traumatic Headache a Potential Role for Glymphatic Pathway Dysfunction Current Pain and Headache Reports 23(9) httpsdoiorg101007 s11916-019-0799-4 [3] Plog B A Dashnaw M L et al (2015) Biomarkers of traumatic injury are transported from brain to blood via the glymphatic system Journal of Neuroscience 35(2) 518-526 httpsdoiorg101523JNEUROSCI3742-142015 [4] Rasmussen M K Mestre H et al (2018) The glymphatic pathway in neurological disorders The Lancet Neurology 17(11) 1016-1024 httpsdoiorg101016S1474-4422(18)30318-1 [5] Reeves B C Karimy J K et al (2020) Glymphatic System Impairment in Alzheimerrsquos Disease and Idiopathic Normal Pressure Hydrocephalus Trends in Molecular Medicine 13-16 httpsdoiorg101016jmolmed201911008 [6] Zhang L Chopp M et al (2019) Role of the glymphatic system in ageing and diabetes mellitus impaired cognitive function Stroke and Vascular Neurology 4(2) 90ndash92 httpsdoiorg101136svn-2018-000203


Usefulness of ultraviolet-induced spectral bone fluorescence

in assessing the time of inhumation

Joanna Tyszer1 Anna Spinek

2 Dorota Bonarska-Kujawa

3

1Faculty of Biology and Animal Science Wrocław University of Environmental and Life Sciences 2Department of Anthropology Polish Academy of Sciences 3Faculty of Life Sciences and Technology Department of Physics and Biophysics Wrocław University of Environmental and Life Sciences

251

Background

Estimating the time since death of an

individual (PMI ndash Post Mortem Interval) is

very important for forensics as well as for

archeology and anthropology Determining

PMI requires an interdisciplinary approach

Therefore creating a simple fast and

effective method of estimating the time

since death can revolutionize both arche-

ology and physical anthropology but also

forensic sciences

The chemical composition of bones changes

with the time they remain in the ground

Differences in bone autofluorescence exci-

ted by ultraviolet radiation can be used to

determine the approximate period of their

inhumation [1 3] This is indicated by the

results of studies carried out in the past by

various researchers but these results are not

unambiguous especially they used different

methods of sample preparation and mea-

surement of bone autofluorescence

The aim of the study was therefore to check

whether it is possible to determine the time

of inhumation of human remains using

spectral analysis of bone fluorescence

excited by ultraviolet radiation


Human bones used for measurements have

been powdered in a mortar They were

small fragments ndash about 05 cm in size taken

from the ribs The bones came from various

periods from the Neolithic to the 16th - 18th

century they were obtained from the

skeletal collection of the Department of

Anthropology Polish Academy of Sciences

Powdered bones were suspended in 09

NaCl solution in a ratio of 1 mg bones 1ml

NaCl solution Measurements were made in

Department of Physics and Biophysics

by spectrofluorimetric method over a wide

range of fluorescence excitation wave-

lengths

Results

The most interesting results were obtained

in the case of bone fluorescence excitation

with 370 and 380 nm wavelengths The

obtained results are presented in figure 1

and figure 2

Differences in fluorescence intensity of

individual samples are clearly visible but

they are not correlated with the chronology

of the periods from which the bones come


Previous studies on dating by bone

autofluorescence also show no relationship

between fluorescence intensity and sample

age [2 3] Instead they indicate a relation-

ship between fluorescence intensity and

collagen content in the bone sample Due to

the varied environmental conditions in which

the bones were found the amount of colla-

gen retained and its degradation products

may differ significantly even between

samples from the same period [4] For this

reason based on the analysis of available

literature and the presented experiment the

only valid conclusion is to carry out more

detailed analyzes on a much larger amount

of material

References [1] Bachmann C Ellis E H Nature vol 208

1328-1331 1965

[2] Hoke N Burger J et al Forensic Science

International vol 228 176e1-176e6 2013 DOI

doiorg101016jforsciint201303013

[3] Capasso L DAnastasio R et al Forensic

Science International vol 272 87-96 2017 DOI


[4] Collins M J Riley M et al Journal

of Archaeological Science vol 22 175-183 1995

DOI doiorg101006jasc19950019

252

Figure 1 Emission spectrum of bones probes with 370 nm excitation wavelength



Sensitive analysis of sarcosine in artificial and real urine

after derivatization with ninhydrin

Dagmar Uhliacuteřovaacute1 Michaela Všetičkovaacute



1

Rene Kizek12

1Department of Research and Development Prevention Medicals sro Tovaacuterniacute 342 742

13 Studeacutenka-Butovice Czech Republic 2Department of Human Pharmacology and

Toxicology VFU Brno Palackeacuteho třiacuteda 19461 612 42 Brno Czech Republic

Background

Sarcosine is an amino acid that is studied as

a potential marker of prostate cancer and

other diseases It is commonly found in

muscle other body tissues and fluids HPLC

with derivatization is commonly used for

sarcosine detection The aim of this work

was to design a simple procedure for sarco-

sine analysis in real urine Sarcosine was

0

10

20

30

40

50

370 390 410 430 450 470 490 510 530 550

Inte

nsi

ty [

au]

Wavelengt [nm]

Excitation 370 nm Złota 21

Groszowice 8

Wrocław - Ołbin 179

Milicz 80

Wrocław - Plac Dominikański 31

Pawłoacutew Trzebnicki 5

Wrocław - Plac Czysty 170

0

10

20

30

40

50

380 400 420 440 460 480 500 520 540 560

Inte

nsi

ty [

au]

Wavelengt [nm]


Groszowice 8


Milicz 80




253

determined by ion exchange liquid chroma-

tography with ninhydrin derivatization


The AAA500 analyzer from Ingos (Prague

Czech Republic) was used for the analyzes

The chemicals hydrindantine thiodiglycol

methylcelsolve and 4 M acetate buffer were

from Ingos (Prague Czech Republic)

Buffer chemicals (mobile phase) such as

citric acid and sodium citrate were from

Lachner (Neratovice Czech Republic)

Ninhydrin sodium chloride and other

chemicals were from Sigma-Aldrich (St

Louis USA) The distilled water was

prepared by the Aqual system (Tisnov

Czech Republic) and the ultrapure water

was prepared by the ELGA system (High

Wycombe United Kingdom) up to 18 MΩ

The pH control was performed using a pH

meter (VWR USA)


It is known that urine is a complex matrix of

ions sugars peptides proteins and other

substances In addition to the significant

qualitative variability is considerable varia-

bility uantitative Amino acids need to be

derivatized to detect them A suitable deri-

vatizing agent is ninhydrin The separated

amino acids react with ninhydrin The

detection solution was prepared from 112

mM ninhydrin 75 methylcellosolve and 1

M acetate buffer Hydrindantine was used as

reducing agent The resulting product was

detected by a two-channel detector at 440

and 570 nm The separation was carried out

in a glass column with an ion exchanger

having a particle size of 8 microm The elution

phase contained 58 mM citric acid 16 mM

sodium citrate 158 mM sodium chloride

and 025 thiodiglycol The mobile phase

flow rate was 025 mlmin and the ninhydrin

flow rate was 02 ml min The reactor

temperature was 131degC The sample inject-

tion volume was 200 microl The optimization

steps included an experiment with samples

in water buffer artificial urine and real

urine When the sarcosine standard in

ultrapure water was used for the assay LOD

17 microM and LOQ 57 microM were detected

When the standard was prepared in dilution

buffer (729 mM citric acid 1968 mM

sodium chloride and 05 thiodiglycol) the

limits were lower (LOD 05 microM and LOQ

18 microM) Using artificial urine (Chutipong-

tande) measurements were performed at pH

6 (untreated solution) and at pH 2 At pH 6

LOD was 60 microM and LOQ was 197 microM

but at pH 2 there was a slight reduction in

the limits to LOD 39 microM and LOQ 129

microM The results indicate a need to adjust the

sample to a low pH The pH 2 was chosen

according to the elution buffer which has a

pH of about 2-22 In mixed urine urine

samples were adjusted to pH 20 21 and

22 Sarcosine calibration at concentrations

of 625 313 156 78 39 and 0 was

performed using mixed urine At pH 20 the

lowest sarcosine detectable concentration

was about 78 microM LOD was 142 microM and

LOQ was 468 microM At pH 21 the lowest

sarcosine detectable concentration was

about 39 microM LOD was 109 microM and LOQ

was 359 microM At pH 22 the lowest sarco-

sine detectable concentration was 156 microM


A typical chromatogram of a real urine

sample with the addition of sarcosine (400

microM) The sample was adjusted to pH 21

with HCl

Conclusions

We have developed a sensitive method for

the detection of sarcosine in ultrapure water

above 2 microM in dilution buffer above 05

microM and in urine sample above 10 microM

of sarcosine The sample optimization method

showed that the highest sarcosine sensitivity

was achieved with a sample at pH of 21

Acknowledgements


of SARKO TEST 1652016

254

References [1] Cernei N Zitka O Ryvolova M Adam V

Masarik M Hubalek J Kizek R (Int J

Electrochem Sci) 7 4286 (2012)

[2] Jiang Y Cheng X Wang C Ma Y (Anal

Chem) 82 9022 (2010)

[3] Shamsipur M Naseri M TBabri M (J Pharm

Biomed Anal) 81-82 65 (2013)


Next-generation sequencing in lung cancer diagnosis and therapeutic

strategies

Katarzyna Wadowska1 Iwona Bil-Lula

1 Mariola Śliwińska-Mossoń

1


Laboratory Haematology Wroclaw Medical University ul Borowska 211A 50-556

Wroclaw Poland

Lung cancer is the leading cause of cancer-

related deaths in the world accounting for

the 25 of cancer mortality 75 of the

patients are diagnosed with lung cancer at

its advanced-stage when treatment options

are limited [1] These alarming reports show

the need of looking for an effective diag-

nostic and therapeutic strategies in early-

stage lung cancer

The last two decades have seen exponential

developments of the genetic and epigenetic

understanding of oncogenic transformation

Lung cancer is the end result of multistage

cancerogenesis with gradually increasing

genetic and epigenetic changes leading to

oncogene activation andor loss of the sup-

pressor gene function [2] Molecular ana-

lysis of these actionable mutations requires

a novel technologies that will become

routine practice in lung cancer diagnosis

In this review we took a look at the most

promising platform in cancer investigetion

ndash next-generation sequencing (NGS)

255

NGS is capable of sequencing millions or

billions of DNA molecules simultaneously

that affords maximal tumor genomic asses-

sment DNA sequencing by NGS includes

whole-genome whole-exome and targeted

sequencing [3 4] Targeted-NGS has a po-

tential to revolutionize clinical diagnosis of

lung cancer through multiplexed detection

of genomic alterations and the analysis of

cancer driver genes for precision cancer

therapy Exemplary gene-panel of somatic

mutations includes BRAF EGFR ERBB2

KRAS NRAS PIK3CA PTEN and TP53

where EGFR and TP53 exhibite the highest

mutation rate and may be a therapeutic

target in lung cancer patients [56]

NGS may detect actionable mutations with

high accuracy and is a promising tool for the

analysis of molecular targets for the initial

diagnosis of disease monitoring of disease

progression and identifying the mechanism

of drug resistance

References

[1] Fred R Hirsch Giorgio V Scagliotti James L

Mulshine Regina Kwon Walter J Curran Yi-Long

Wu Luis Paz-Ares Lung Cancer Current

Therapies and New Targeted Treatments Lancet

2017 Jan 21389(10066)299-311

[2] Potempa M Jonczyk P Zalewska-Ziob M

Molekularne uwarunkowania raka płuca Onkol

Prak Klin 2014104199-211

[3] Zhang X Liang Z Wang S Lu S Song Y

Cheng Y Ying J Liu W Hou Y Li Y Liu Y Hou

J Liu X Shao J Tai Y Wang Z Fu L Li H Zhou

X Bai H Wang M Lu Y Yang J Zhong W Zhou

Q Yang X Wang J Huang C Liu X Zhou X

Zhang S Tian H Chen Y Ren R Liao N Wu C

Zhu Z Pan H Gu Y Wang L Liu Y Zhang S Liu

T Chen G Shao Z Xu B Zhang Q Xu R Shen L

Wu Y Tumor Biomarker Committee

OBOCSOCOC Application of next-generation

sequencing technology to precision medicine in

cancer joint consensus of the Tumor Biomarker

Committee of the Chinese Society of Clinical

Oncology Cancer Biol Med 2019 Feb16(1)189-

204 doi 1020892jissn2095-394120180142

PMID 31119060 PMCID PMC6528448

[4] Serratigrave S De Summa S Pilato B Petriella D

Lacalamita R Tommasi S Pinto R Next-generation

sequencing advances and applications in cancer

diagnosis Onco Targets Ther 2016 Dec 297355-

7365 doi 102147OTTS99807 PMID 27980425

PMCID PMC5144906

[5] Sim WC Loh CH Toh GL Lim CW Chopra

A Chang AYC Goh LL Non-Invasive Detection of

Actionable Mutations in Advanced Non-Small Cell

Lung Cancer Using Targeted Sequencing of

Circulating Tumor DNA Lung Cancer 2018

Oct124154-159 doi

101016jlungcan201808007 Epub 2018 Aug 8

[6] Xu X Yang Y Li H Chen Z Jiang G Fei K

Assessment of the Clinical Application of Deteting

EGFR KRAS PIK3CA and BRAF Mutations in

Patients with Non-Small Cell Lung Cancer Using

Next-Generation Sequencing Scand J Clin Lab

Invest 2016 Sep76(5)386-92 doi 101080

0036551320161183813 Epub 2016 May 23


Innovative liposomes systems as transporters of active cargo

E Waglewska1 U Bazylińska

1



Despite the continuous development of

medicine the application of many actives

substances are limited It is associated with

their low water solubility potential side

effects and unfavorable pharmacokinetics

For this reason the nanocarriers are very

important structures that are used to create

effective and at the same time safe drug

delivery systems Currently liposomes are

the most intensively studied delivery

systems of active substances It is connected

with their numerous properties such as

256

biocompatible non-toxicity and a very good

ability to encapsulate hydrophilic and hyd-

rophobic ingredients Moreover the lipo-

somes are composed of natural components

of biological membranes ndash phospholipids

what give them better biocompatibility and

biodegradability [1]

One of the key parameter in designing nano-

scopic drug delivery systems is the size of

liposomes which influence their properties

and application The smaller nanocarriers

are less captured by mononuclear phagocyte

system (MPS) cells They have also impro-

ved accumulation in cancerous tissues

which is connected with the occurrence of

the enhanced permeability and retention

(EPR) effect Liposomes of various sizes

can be obtained by utilization of different

techniques The main popular way to create

multilamellar liposomes MLV (above 1 microm)

is the thin-film hydration method The

received MLV dispersion is submitted to

sonication or extrusion in order to obtain the

smaller nanocarriers (lt 200 nm) [2]

The therapeutic index of biologically active

substances encapsulated in liposomes is

higher in comparison to drugs transported in

free form This is due to reduction of the

exposure of healthy tissues to the encap-

sulated drugs Furthermore functionalize-

tion and numerous surface modifications of

vesicles permit to obtain stable liposomes

which can allow transport drugs to the target

tissues It is also possible to control the

release of active cargo due to the action of

a specific factor (eg temperature pH ultra-

sound magnetic field or laser irradiation)

[3] Different kind of modified liposomes

may contribute to development of the inno-

vative and effective treatments for many

diseases (including cancer) Due to that fact

there is a huge need to conduct studies in

this field

Acknowledgements






References [1] G Bozzuto A Molinari International Journal

of Nanomedicine vol 10 975-999 2015 DOI

102147IJNS68861

[2] TOB Olusanya RR Haj Ahmad et al

Molecules vol 23 1-17 2018 DOI 103390

molecules23040907

[3] MK Riaz MA Riaz et al International

Journal of Molecular Sciences vol 19 1-27 2018

DOI 103390ijms19010195


Modulation of blood-brain barrier permeability by activating

adenosine A2 receptors in oncological treatment

Kamila Wala1 Julita Kulbacka

2


and Cellular Biology Faculty of Pharmacy Wroclaw Medical University

The blood-brain barrier (BBB) plays an

important protective role in the central

nervous system and maintains its home-

ostasis It regulates transport into brain

tissue as well as protects neurons against the

toxic effects of endo- and exogenous

substances circulating in the blood thus

providing proper functioning of the central

nervous system [1]

However in case of neurological diseases or

primary brain tumors ie gliomas the

higher permeability of the blood-derived

substances in the brain tissue is necessary

Modulation of the blood-brain barrier

257

permeability may contribute to an increase

in the concentration of the drug in the CNS

and thus increase the effectiveness of

therapy

Currently applied methods of treatment for

the primary brain neoplasms include surgi-

cal tumor removal radiation therapy and

chemotherapy Despite the above-mentio-

ned treatment methods the prognosis of

primary brain tumors still remains bad (for

instance malignant glioma median survival

is less than 12 months) [2] Moreover

chemotherapy options seem to be limited

due to low drug penetration into the

cancerous tissue Therefore further research

is required to increase therapeutic options

in patients with brain tumors The aim of the

article is to assess the possibility to increase

the BBB permeability to increase the

effectiveness of oncological therapy

In this case the Adenosine 2A receptor

(A2AR) seems to be a promising therapy-

target due to its important role in the

modulation of BBB The action of A2A

agonists increases the permeability of the

blood-brain barrier by actin-cytoskeletal re-

organization and acting on the tight junc-

tions [3] Interestingly it has been proven

that the gene encoding this receptor is

overexpressed in the tumor area Moreover

clinical trials using the chemotherapy agent

(Image-guided paclitaxel injection) together

with an A2A nano-agonist showed a better

antitumor effect and prolonged survival [4]

Adenosine 2A receptor modulation may be

a potential target to increase the effectivity

of chemotherapeutics and to improve the

results of cancer therapy

Acknowledgments The work was created

as part of the activity of the Student

Research Group Biology of Cancer Cell at

the Wroclaw Medical University (SKN No

K 148) and Statutory Funds of Department

of Molecular and Cellular Biology No

SUBD26020009

References

[1] Y Zhou Z Peng et al Journal of Controlled

Release vol 270 290-303 2018 DOI 101016

jjconrel201712015

[2] FB Furnari T Fenton et al Genes amp

Development vol 21 2683-2710 2007

DOI101101gad1596707

[3] DG Kim MS Bynoe Molecular Neuro-

biology vol 521 664-678 2015 DOI101007

s12035-014-8879-2

[4] X Gao Q Yue et al Theranostics vol 811

3126 2018 DOI 107150thno24784


The influence of disiloxane derivatives on activity and expression

of ABCB1 transporter in colon cancer cells

Olga Wesołowska1 Kamila Środa-Pomianek

1 Maria Błaszczyk

1 Joseph Molnar

2

Krystyna Michalak1

1Department of Biophysics Wroclaw Medical University 2Institute of Medical Microbiology

and Immunobiology University of Szeged

Background

Since chemotherapy continues to be

a method of choice for the treatment of

cancer any factors that undermine its

effectiveness constitute a serious therapeutic

issue In majority of patients the initial

response to chemotherapy is satisfactory

however the consequent occurrence of mul-

tidrug resistance (MDR) results in a deve-

lopment of progressive disease Among

many mechanisms that may lead to MDR

the overexpression of ATP Binding Cassette

(ABC) transporters such as ABCB1 protein

258

(P-glycoprotein MDR1) seems to be the

most important ABCB1 is a transporter that

utilizes energy gained from hydrolysis of

ATP to pump many structurally variable

substrates (including anticancer drugs) out

of the cell [1] The most basic idea to over-

come MDR is to use the inhibitor of trans-

porter (MDR modulator) along with chemo-

therapy in hope to increase intracellular

accumulation of an anticancer drug and to

improve the final outcome of the treatment


The pair of human colon cancer cell lines ndash

sensitive and resistant to doxorubicin (LoVo

and LovoDx) as well as MDCK calls

transfected with human ABCB1 gene have

been employed as a model system to study

the influence of putative MDR modulators

on ABCB1 expression and transport acti-

vity Cytotoxicity of the modulators was

measured via SRB assay Isobolographic

analysis was employed to study putative

synergism between doxorubicin and modu-

lators Accumulation of ABCB1 substrates

was monitored by functional tests based on

intracellular fluorescence measurement as

well as by fluorescence microscopy Protein

expression level was assessed by Western

blot

Results

Two disiloxane derivatives were relatively

cytotoxic both to colon cancer cells and to

MDCK cells (recorded IC50 values were

below 25 microM) Each compound was tested

as a putative MDR modulator in concen-

tration ca 10 times lower than its IC50

value Both derivatives were demonstrated

to increase the sensitivity of LoVoDx cells

to doxorubicin The existence of synergism

between doxorubicin and the studied modu-

lators was also demonstrated Additionally

the increased accumulation of anticancer

drug within resistant cells was demonstrated

in the presence of the studied derivatives

The spatial arrangement of doxorubicin was

affected by them too The inhibition of

transport activity of ABCB1 protein

by disiloxane derivatives was observed both

in LoVoDx cells and in MDCK cells

overexpressing human ABCB1 transporter

Rhodamine 123 was used as fluorescent

substrate analogue Its accumulation was

significantly increased in both types of cells

treated by the modulators Moreover both

studied derivatives strongly reduced

ABCB1 protein expression in doxorubicin-

resistant colon cancer cells


Two studied disiloxane derivatives were

demonstrated to be effective MDR modula-

tors in doxorubicin-resistant colon cancer

cells Used in in concentrations in which

they were non-toxic to the cells they were

able to increase sensitivity of resistant cells

to anticancer drug Two processes seemed

to be responsible for MDR-reversal activity

of the studied compounds They both

inhibited transport activity of ABCB1 and

decreased the expression of this transporter

References [1] FJ Sharom Pharmacogenomics vol 9 105-

127 2008 DOI 1022171462241691105


Morphological changes in the ovarian cortex in the early stages of

diabetes mellitus in conditions of chronic stress

Wiatr M1 Bagaylyuk LB

2 Zhurakivskyi VM

3 Miskiv VA

4 Bodnarchuk YuV

5

Ivantsiv OR2

1Faculty of Foreign citizensrsquo Training Department Ivano-Frankivsk National Medical

University Ukraine 2Department of Clinical Anatomy and Operative Surgery Ivano-

259

Frankivsk national medical university Ukraine 3Department of Midwifery and Gynaecology

named after professor Lanovyi ID Ivano-Frankivsk National Medical University Ukraine 4Department of Human Anatomy Ivano-Frankivsk National Medical University Ukraine 5Department of Pharmacology Ivano-Frankivsk National Medical University Ukraine

Background

In clinical and experimental studies much

attention has been paid to the problem of

diabetic polyendocrinopathy which leads to

an impaired female reproductive function

[2] Evidence from the scientific literature

indicates at the developing of the erased

forms of dysgenesis of the gonads in dia-

betes mellitus (DM) which can further

cause menstrual disorders in particular

leading to a secondary amenorrhea of ova-

rian genesis which is confirmed by cases of

DM in girls Therefore the purpose of our

research was to study the histo-ultrastruc-

tural remodeling of the ovarian cortex on

the 14th day of streptozotocin-induced

diabetes mellitus (SDM) in conditions of

chronic stress


The study used 20 adult white female rats

(weighing 180-200 g) which were equally

divided into 4 groups 1st group with

modeled SDM and chronic immobilization

stress 2nd group with SDM 3rd group with

chronic immobilization stress 4th group -

intact animals The SDM was simulated by

a single intraperitoneal administration of

streptozotocin SIGMA (USA) (6 mg per

100 g of body weight) which was diluted in

01 M citrate buffer with a pH of 45

Chronic immobilization stress was perfor-

med by placing the animal in a sealed

plastic container for 5 hours a day Material

sampling was carried out on the 14th day of

experiment Histological electron micros-

copic biochemical and statical methods

were used The study was approved by

bioethics commitee

Results

On the 14th day of experiment the level


group of rats is the highest compared with

4th group and is 1561plusmn223 mmoll

(plt0001) and 721plusmn072 (plt001) respec-

tively in 2nd group ndash 1353plusmn213 mmoll

(plt0001) and 612plusmn048 (plt001) in 3rd

group ndash 545plusmn073 mmoll (pgt005) and


animals the above indicators are 435plusmn052



indicate the development of decompensated

DM

On the background of hyperglycemia in the

1st and 2nd study groups in comparison

with the 4th group of animals quantitative

and qualitative changes of different ovarian

follicles were observed In particular the

number of primordial follicles decreased by

132 ndash 112 (plt005) primary ndash by 303

ndash 224 (plt001) maturing ndash by 35- 29

while the number of atressive follicles

increases in 15-20 times (plt005) In the

eggs of ovaries of the 1st and 2nd groups of

animals during this period we observe the

ultrastructural changes of the follicular

epitheliocytes the electron-optical density

of the cytoplasm increases the configu-

ration of the nucleus changes the chromatin

shrinks into the lumps the mitochondrial

matrix is enlightened some of their cristae

are destroyed the cisternae of granular

endoplasmic reticulum are enlarged the

amount of attached ribosomes on their

surface is decreased The same changes are

observed in endocrinocytes of the inner

folder In general in the field of view of the

examined ovaries the number of maturing

follicles is decreased Such changes occur

260

on the background of arterial spasm and

dilation of the venous units of the hemo-

microcirculatory blood flow In the 3rd

group of rats in comparison with the 4th

group no significant quantitative and quail-

tative changes of the follicles were obser-

ved whereas in the vessels of the hemo-

microcirculatory blood flow we observed

a spasm of arterioles and an increased

amount of micropinocytotic vesicles in

endotheliocytes of microvessels


The changes we detected in the cortex of the

ovaries of the 1st and 2nd groups of animals

can be considered as a serious deviation of

the organogenesis of the reproductive

system the occurrence of which is asso-

ciated with an impaired regulation of folli-

culogenesis in particular with an increased

blood glucose level We have found a large

number of atresive follicles the presence of

which in DM was noted by a number of

authors (Demidov VN Adamian LV et al

2006) Insulin plays an important role in the

pathogenesis of polycystic ovary syndrome

(POS) both directly and indirectly Insulin

has a direct stimulating effect on the sec-

retion of androgens by the ovarian theca

cells and enhancement of lutein-dependent

synthesis of the androstenedione by the

theca and the ovarian stromal cells The

reduction of epimerase activity is consi-

dered as one of the factors in the formation

of polycystic ovary syndrome which occurs

in diabetes (Heimark D McAllister J

Larner J 2014) Another factor that leads to

a decrease in the number of maturing folli-

cles is hypoxia due to the development of

diabetic microangiopathy [1 3]

Thus SDM and its combination with chro-

nic immobilization stress lead to a decrease

in the number of primordial follicles on the

background of the increased number of

atresive follicles which can be considered

as a precondition for the formation of infer-

tility andor early beginning of menopause

the exhausted ovary syndrome in DM

References [1] F Cosentino PJ Grant et al 2019 ESC

guidelines on diabetes pre-diabetes and

cardiovascular diseases developed in collaboration

with the EASD European Heart Journal 1 69

2019

[2] IM Kapshuk HO Islamova Osoblyvosti

likuvannia syndromu polikistoznykh yaiechnykiv u

zhinok z metabolichnym syndromom Slovo pro

zdorovia Endokrynna hinekolohiia 13 26 2018

[3] R Madonna CR Balistreri et al Diabetic


therapeutic approaches Vascul Pharmacol 90 1-7

2017 doi 101016jvph201701004


Study of interaction tyrosine kinase inhibitors with human serum

albumin (HSA) by different spectroscopic techniques

Katarzyna Wiglusz Igor Mucha Maciej Nawaryński and Rafal J Wiglusz 1Faculty of Pharmacy Wroclaw Medical University Borowska 211 A 50-566 Wroclaw

Poland 2Institute of Low Temperature and Structure Research Polish Academy of Sciences

Okolna 2 50-422 Wroclaw Poland

Background

Tyrosine kinase inhibitors including ima-

tinib mesylate andor ibrutinib are com-

monly used for combination chemotherapy

0[1] In the case of these drugs that are

characterized by high plasma protein bin-

ding and can affect pharmacokinetic

parameters the potency of drugs as well

as their toxicity The aim of the study was to

examine the interaction ibrutinib and

261

imatinib mesylate with HSA in vitro at

different temperatures (25-37 oC) at pH 74

In our study the drugs-albumin complex

formation were investigated by fluores-

cence UV-vis absorption and circular

dichroism spectroscopy


HSA was purchased from Sigma Chemical

Co 97 Imatinib mesylate (97) and

ibrutinib (96) were obtained from Abcr

GmbH Germany HSA solutions were

prepared in the phosphate buffer solutions

(p-H 74) and stored in the dark at 4degC

UV-Vis absorption spectra were carried out

using a AnalyticJena UV-Visible spectro-

photometer Specord 50 plus Fluorescence

measurements were performed on a Jasco

FP-8200 spectrophotometer Circular di-

chroism measurements were recorded on

a Jasco J-1500 spectropolarimeter

Results

We observed strong quenching of the fluo-

rescence at 280 nm excitation wavelength

what clearly indicates that the binding of the

tyrosine kinase inhibitors to HSA changed

the microenviroment of tryptophan residue

and the tertiary structure of HSA It has

been found that tyrosine kinase inhibitors

(imatinib mesylate and ibrutinib) bound

to albumin with the binding affinity

constants (~105 M-1 at pH 74) Moreover it

has been noted that the hydrophobic forces

and hydrogen bonding play major roles in

the complex formation Analysis of circular

dichroism spectra has shown that the

α-helical structure of albumin decreased

about 7 after addition of the anticancer

drugs


The affinity of the tyrosine kinase inhibitors

to albumin was slightly increase in presence

both drugs Interestingly the effect of

ibrutinibe on HSA binding was stronger

than imatinibe mesylate This work can

provide a useful information related to an

interaction of the tyrosine kinase inhibitors

with the albumin and help to understand the

influence of both drugs to the albumin

binding

References 1 LK Shea FM Mikhail A ForeroTorres RS

Davis Concomitant imatinib and ibrutinib in

a patient with chronic Clin Case Rep 2017 5(6)

899-901


262

Activation of blood platelets in chemical ischemia and reperfusion

conditions ndash preliminary research

Kornela Wiśniewska1 Alina Rak-Pasikowska

1 Aneta Wrzyszcz

1 Iwona Bil-Lula

1

1Division of Clinical Chemistry and Laboratory Hematology Department of Medical

Laboratory Diagnostics Faculty of Pharmacy Wroclaw Medical University

Background

Platelets activation leads to changes in the

platelets shape release the content of α and

β granules and increase expression of nume-

rous proteins on the platelets surface among

others P-selectin activated glycoprotein

IIbIIIa (PAC-1) or lysosomal associated

membrane protein (CD63) Physiologically

platelet activation occurs when the blood

vessel is damaged However increased

platelet activation may be the result of

numerous processes such as inflammation

or atherosclerosis and concurrent ischemia

increased concentrations of reactive oxygen

species (ROS) and platelet agonists Platelet

functions and mechanisms of activation in

diseased states are not as well studied

as in normal healthy conditions and may not

be the same The aim of the study was to

investigate whether platelets activated under

chemical ischemia and reperfusion condi-

tions in vitro model


Citrated venous blood samples collected

from 5 healthy human volunteers was the

test material Platelets were isolated on

a continuous density gradient OptiPrep

(Sigma-Aldrich) by method developed in

our Department Isolated platelets under-

went a chemical ischemia and reperfusion

(IR) procedure consisted of three stages 15

minutes stabilization 9 minutes ischemia

and 10 or 20 minutes reperfusion ndash protocol

presented on figure 1 In the control series

all stages were performed in the same way

as in experimental series but ischemia buffer

was replaced by stabilization buffer Plate-

lets were stabilized and reperfused in

HEPES buffer with the addition of 55

μmolL CaCl2 and 075 mgmL BSA

Ischemia was achieved by the addition of

40 mmolL sodium cyanide ndash an oxygen

respiration inhibitor and 44 mmolL

2-deoxyglucose ndash a glycolysis inhibitor

Activation of platelets was assessed by flow

cytometry after each stage of the procedure

using CyFlow cytometer (Sysmex) Three

markers of platelet activation were evalu-

ated in the study P-selectin PAC-1 and

CD63

Results

A continuous increase in PAC-1 expression

was observed at each subsequent stage of

the procedure in both the experimental and

control series However a significant incre-

ase was observed after the last stage by 18

in experimental series (plt005) and 25 in

control series (plt005) compared to first

stage

In the case of P-selectin an maximal incre-

ase in platelet surface expression was

observed after the hypoxia stage compared

to stabilization stage (plt005) in the experi-

mental series then decreased rapidly In

control series maximal increase was obser-

ved after 10rsquo reperfusion and gently decre-

ased The largest difference in P-selectin

expression between experimental and cont-

rol series was observed after 10rsquo reperfusion

(p=0043)

After 10rsquo reperfusion in the experimental

group the CD63 expression was on the

highest level ndash the number of labeled plates

is on average 9 higher than at the stabili-

zation stage (plt005) and almost 3 higher

than in the control group at the same stage

263

After 20 minutes of reperfusion CD 63

expression was the same in both series


Platelets gets activation in diseases such as

myocardial infarction or stroke in which in

addition to ischemia an inflammatory com-

ponent is present [12] It is considered that

platelet activation occurs in sites with

increased levels of reactive oxygen species

[3] On the other hand the degree of platelet

activation was similar in ischemic and

nonischemic patient with heart failure [4]

The presented results of expression of the

three platelets activation markers PAC-1

P-selectin CD63 serve the evidence of pla-

telet activation under chemical ischemia and

reperfusion however requires further

experiments and perhaps create a model that

will allow to reduce the test time and the

impact of other factors potentially acti-

vating the platelets like centrifugation

References [1] Cherian P Graeme J et al Stroke Vol342132-

7 2003 DOI 10116101STR0000086466

32421F4

[2] Cameron SJ Ture SK et al Circulation

vol13247-58 2015 DOI 101161

CIRCULATIONAHA115015656

[3] Smyth SS McEver RP et al J Thromb

Haemost vol 71759-66 2009 DOI 101111

j1538-7836200903586x

[4] Gurbel PA Gattis WA et al Am Heart J vol

1431068-75 2002 DOI 101067mhj2002

121261

Figure 1 Scheme of experimental protocol


Safety and effectiveness of aspartame use in body weight reduction

Adam Wojcieszonek Natalia Komorniak Viktoria Hawryłkowicz

Department of Human Nutrition and Metabolomics Pomeranian Medical University

in Szczecin Poland

Background Excessive body weight is a growing problem in both developed and still deve-loping countries One of the main causes of obesity is the high consumption of products rich in easily absorbed carbohydrates

Among people who use a low-calorie diet a phenomenon of a strong need to eat sweet food which is source of condensed content of sucrose or glucose-fructose syrup can be observed Therefore sweet products with lower energy value are becoming more and more popular nowadays The composition

264

of these products is largely based on low calorie sweeteners One of the most exa-mined is aspartame Aspartame has gained immense popularity because of its sweet-ness closest to sucrose The caloric content of aspartame is 4 kcalg which is the equivalent to the sucrose caloric content However 1g of aspartame is about 200 times sweeter than sucrose This sweetener is a dipeptide which is broken down into aspartic acid phenylalanine and methanol in the human body In the next stage methanol is oxidized to formaldehyde and formic acid Both of these compounds are toxic to the human body

The aim of the study was to evaluate that aspartame can be used as an alternative to sucrose in the quest to reduce excessive weight

Material and Methods A review of the literature present in the Pubmed database from 2005-2020 was conducted

Results Literature to date reports that aspartame is a carcinogen capable of causing malignant lesions at a lower dose level than current acceptable human daily intake (EFSA) However these studies are being under-mined by EFSA researchers claiming that due to the high percentage of spontaneous cancers cancer diagnosis problems and con-cerns about the impact of chronic infections far-reaching conclusions cannot be drawn from this study EFSA has set the Accep-table Daily Intake (ADI) value of aspartame to be 40mg kg bw Studies also showed that aspartame has no effect on the intestinal microbiota It is very quickly hydrolysed in the small intestine and absorbed there In fact it doesnt even reach to the large intes-tine What is very interesting ndash study that compared the consumption of light drinks with water showed that people on a diet and consuming light drinks achieved signify-cantly greater weight reduction (approx

124 kg) compared to people consuming only water

Discussion and conclusions Most studies proving the high harmfulness of aspartame have been conducted in rats which is not a reliable reference to the human body However it is indisputable that the methanol formed after decom-position from aspartame is toxic to the human body However it is worth noting that the EFI ADI value for aspartame (40mg kg mc) is very difficult to eat during the day because as a society we do not appro-ach this value even considering the risk group of children One litre of light cola contains 529mg of aspartame an 80-kilo person would have to drink 55 litres of drink a day to cross the limit set by EFSA By drinking the same amount of classic cola 583g of sugar would be supplied in this way introducing an additional 2332kcal into the diet The potential good effects of aspartame in weight loss may be because of phenomenon that people who consume light drinks is less willing to reach for high calorie snacks

Based on the analysed literature it can be concluded that the use of aspartame as a replacement for sucrose may help in reducing excess weight

Undoubtedly it is necessary to conduct further research in order to unequivocally define aspartame as a completely safe substance

References [1] K A Higgins i R D Mattes A randomized controlled trial contrasting the effects of 4 low-calorie sweeteners and sucrose on body weight in adults with overweight or obesity Am J Clin Nutr t 109 nr 5 s 1288-1301 2019 doi 101093ajcnnqy381 [2] F J Ruiz-Ojeda J Plaza-Diacuteaz Effects of Sweeteners on the Gut Microbiota A Review of Experimental Studies and Clinical Trials Adv Nutr Bethesda Md t 10 nr suppl_1 s S31-S48 2019 doi 101093advancesnmy037

265

[3] P J Rogers i in Does low-energy sweetener consumption affect energy intake and body weight A systematic review including meta-analyses of the evidence from human and animal studies Int J Obes 2005 t 40 nr 3 s 381-394 mar 2016 doi 101038ijo2015177 [4] B A Magnuson i in Aspartame A Safety Evaluation Based on Current Use Levels

Regulations and Toxicological and Epidemiological Studies Crit Rev Toxicol t 37 nr 8 s 629-727 2007 doi 1010801040844070 1516184 [5] Chattopadhyay U Raychaudhuri i R Chakraborty Artificial sweeteners ndash a review J Food Sci Technol t 51 nr 4 s 611-621 2014 doi 101007s13197-011-0571-1


Biological evaluation of new synthesized pyrimidine derivatives

Aleksandra Wolska1 Aleksandra Mikołajczyk

1 Marcin Stolarczyk

2 Agnieszka

Matera-Witkiewicz1

1Screening Laboratory of Biological Activity Tests and Collection of Biological Material Faculty of Pharmacy Wroclaw Medical University 211A Borowska 50-556 Wrocław Poland 2Department of Organic Chemistry Faculty of Pharmacy Wroclaw Medical University 211A Borowska 50-556 Wrocław Poland

Background Most recent data indicate cancer to be the second leading cause of death in the world Since the widely applied chemotherapy has many side effects it is important to develop new drugs to determine more effective therapy The compounds with pyrimidine core reveal many therapeutic activities eg antimicrobial [1] anti-inflammatory [2] and antioxidant [3] Moreover pyrimidine deri-vatives have been widely tested for their antitumor activity [4 5] In our lab a series of pyrimidine derivatives were synthetized and evaluated for anticancer effects

Material and Methods The synthesized compounds were tested for their cytotoxic activity on following cell lines L-929 (Mouse C3HAn connective tissue) A172 (Human glioblastoma) AGS (Human Caucasian gastric adenocarci-noma) HepaRG (Human hepatoma cell) and HeLa (Human cervix epitheloid carci-noma) Neutral Red uptake assay was per-formed for both neoplastic and non-neo-plastic cell lines according to the protocol [6] Staining with FDAPI and flow cyto-metry were performed with modification based on protocol [7] to confirm results from primary screening Further cells were

stained with Hoechst 33342 according to the manufacturerrsquos instruction and examined under fluorescence microscope

Results Cytotoxicity was primary tested on non-neoplastic L929 cell line Pub-1 has significantly decreased the cell viability (47 for 1mM) Pub-2 and Pub-3 have not exhibited cytotoxic activity (Pub-2 96 and Pub-3 112 for 1mM) Neutral red assay was further used to investigate the proli-ferative inhibition for neoplastic cell lines respectively Compound concentration applied on cells were in the range 10-1000 microM The IC50 was calculated for Pub-2 on HepaRG and for Pub-3 on AGS HepaRG HeLa and A172 (Tab 1)

Table 1 IC50 calculated from Neutral Red test results after 72-hours exposure with Pub-2 and Pub-3

HepaRG AGS A172 HeLa

Pub-2 4042 microM gt1000 microM

gt1000microM

gt1000 microM

Pub-3 3127 microM 5024 microM 5262 microM 3554 microM

Flow cytometry analysis were performed on

AGS cell line using Fluorescein Diacetate

and Propidium Iodide dyes to reveal the

viable and non-viable cells The IC50

266

calculated from flow cytometry confirmed

results obtained by primary screening (IC50

5302 microM)

Changes in cell and nucleus morphology

were observed under fluorescence micro-

scope for AGS Cells exposed on Pub-3 in

IC50 concentration were stained with

Hoechst 33342 Results have been shown

on Fig 1 The apoptotic bodies have been

marked with the arrows


The screening test results show cytotoxic

activity on cancerous cell lines for Pub-2

and Pub-3 and their lack of interaction with

non-neoplastic cells Pub-3 has broad spec-

trum of activity causing the viability decre-

ase for four cell lines The most potent

toxicity reveal against stomach cancer cells

Further analysis of molecular mechanism

are required especially for Pub-3 on AGS

cell line due to potential therapeutic usage

The research was founded by Wroclaw

Medical University (project number

SUBD25019012)

References [1] S Nag R Pathak et al Bioorg amp Med Chem

Letter 16 3824-3828 2006 101016jbmcl

200604020

[2] HE Elsherbiny SM Khaled Polycyclic

Aromatic Comp 1-17 2019 10108010406638

20191653941

[3] AA Abu-Hashem MF El-Shehry Eur J Med

Chem vol 44 (11) 4557-4566 2009 101016

jejmech200906024

[4] Naresh Kumar R Malla Reddy G et al

Journal of Heterocyclic Chemistry 101002

jhet1833

[5] Fouad MM El-Bendary ER et al Bioorganic

Chemistry 2018 101016jbioorg201809022

[6] G Repetto A del Peso et al Nature Protocols

vol 3(7) 1125-1131 2008 101038nprot200875

[7] KH Jones JA Senft J Histochem amp Cytochem

3377 1985 1011773312578146

Figure 1 Morphological changes of AGS cell line after Pub-3 treatment

Aa ndash Cell characteristics in control (medium with 1 DMSO) Bb Cells after exposure


267

The effect of anticancer palladium(II) complex on erythrocyte

properties ndash in vitro studies

Magdalena Worobiec1 Aleksandra Włoch

2 Hanna Pruchnik

2

1Student of Faculty of Biology and Animal Science Wrocław University of Environmental

and Life Sciences 2Faculty of Life Sciences and Technology Department of Physics

and Biophysics Wrocław University of Environmental and Life Sciences

Background

Cancers are one of the main causes of the

premature death of people in the world so

the search for new solutions in the fight

against this disease is necessary and a big

challenge Chemotherapy remains one of

the methods of treatment but unfortunately

most cytotoxic compounds used in the

therapy are not only toxic to cancer cells but

cause numerous side effects (neurotoxicity

myelosuppression nephrotosis) Drug resis-

tance is also a problem It is therefore reaso-

nable to synthesize and test new compounds

with potential anti-tumor activity which will

exhibit less toxicity to normal cells

Our previous research shows that the palla-

dium(II) complex with tris(2-carboxyethyl)

phosphine ndash trans-[PdCl2(TCEP)2] (Fig1)

has anti-tumor activity against many cell

lines eg SK-mel (malignant melanoma)

SH-4 (melanotic melanoma) Colo-829

(malignant melanoma) and C-32 (amela-

notic melanoma) For some of them eg

MDA-MB-231 (triple negative breast

cancer) the activity of PdTCEP (IC50 =

8167) is much higher than activity of cis-

platinum (IC50 = 6368) [1]

The aim of the study was to examine the

toxicity of PdTCEP against normal cells by

measuring the hemolytic activity of the

palladium complex and investigating the

effect of PdTCEP on the physicochemical

parameters of the biological membrane The

same tests were performed also for tris(2-

carboxyethyl)phosphine

P(CH2CH2COOH)3 (TCEP)

It is worth emphasizing that the biological

activity of palladium(II) compounds with

tris(2-carboxyethyl) phosphine has not been

tested before


The object of our research were erythrocyte

cells erythrocyte lipid-protein membranes

(shadows) and lipid membranes (monolayer

liposomes formed from egg phosphatidyl-

choline)

Hemolytic activity was determined

by spectrophotometric method while the

effect of PdTCEP and TCEP on biophysical

properties (fluidity hydration degree of

ordering) of membranes was investigated

using fluorimetric method (using Prodan

Laurdan and DPH) The effect of test com-

pounds on erythrocyte cell shapes was

analyzed using an optical microscope In

order to approximate the molecular mecha-

nism of interaction of PdTCEP and TCEP

with the biological membranes measure-

ments were made using infrared spectro-

scopy (ATR-IR) of lipid-protein erythrocyte

membranes modified with test compounds

Results

It was found that in a wide range of concen-

trations (up to 200 M) both PdTCEP and

TCEP do not cause hemolysis but affect the

shape of erythrocytes After adding invest-

tigated compounds red blood cells transfor-

med from a dual concave disks (discocytes)

into echinocytes which may suggest that

PdTCEP and TCEP are present in the outer

lipid monolayer they increase the surface

relative to the inner layer causing a change

in the curvature of the bilayer which leads

268

to deformation of membranes and appen-

dages [2] PdTCEP practically did not affect

the fluidity of the hydrophobic part of the

bilayer while TCEP caused its slight decre-

ase Both compounds contributed to a slight

decrease in the degree of ordering of the

hydrophilic part of the lipid bilayer so they

most likely interact only with the polar part

of the cell membrane This is confirmed by

the results of IR spectra changes are

observed mainly in the vibration band of the

polar phosholipids of lipid-protein mem-

brane of erythrocytes


To sum up PdTCEP in a wide concen-

tration range is not toxic to erythrocytes

does not cause damage to the cell mem-

brane practically does not change its flui-

dity PdTCEP therefore appears to be

a promising compound for further research

of the mechanism of antitumor activity

References [1] H Pruchnik T Lis et al Journal of Inorganic

Biochemistry vol 156124-129 2016 doiorg

101016jjinorgbio201512001

[2] A Kozubek M Stasiuk et al 2009 Postępy

Biochemii vol 55425-433 2009



Biophysics of Wrocław University of

Environmental and Life Sciences

Fig 1 Molecular structure of palladium(II) complex with tris(2-carboxyethyl)phosphine

ndash PdTCEP [1]


Synthesis structures and in silico studies of disubstituted 5-methyl-7-

phenylpyrido[34-d]pyridazine derivatives

Anna Woacutejcicka1 Anna Pyra

2 Lilianna Becan

1 Iwona Bryndal

1

1Department of Drugs Technology Wroclaw Medical University Borowska 211 50-556

Wroclaw 2Faculty of Chemistry University of Wroclaw F Joliot-Curie 14 50-383 Wroclaw

269

Background

Biological investigations have shown that

compounds containing the pyrido[34-d]py-

ridazine scaffold have a broad spectrum of

activity Anticancer analgesic diuretic

antimicrobial activities were found [1] The

various biological properties of pyrido[34-

d]pyridazine derivatives are the main reason

for the preparation of new compounds

containing this scaffold


The chemicals for the syntheses were pur-

chased from Chempur and Sigma-Aldrich

Studied compounds were synthesized ac-

cording to the method presented before [2]

Structures of the newly synthesized com-

pounds were confirmed by FTIR 1H NMR 13C NMR and MS spectra The crystals of

some of compounds of the series of studied

derivatives were obtained X-ray crystallo-

graphy confirms their chemical structure

Results

The aim of this study was the synthesis of

new NN- or NO-substituted 5-methyl-7-


with potential biological activity The pre-

viously obtained 5-methyl-7-phenyl-23-

dihydropyrido[34-d]pyridazine-14-dione

was alkylated to the corresponding alkilaryl

derivatives An in silico analysis of the

bioavailability and potential pharmacolo-

gical activity of the obtained compounds

was performed The results of the analysis

showed that the received compounds could

possess biological activity and satisfactory

absorption through biological membranes

and after oral administration


The starting compound 5-methyl-7-phenyl-

23-dihydropyrido[34-d]pyridazine-14-dione

may exists in two tautomeric forms 1-oxo

and 1-hydroxy denoted as (1) and (2) in

Scheme below In this way NN- or NO-

disubstituted derivatives may form in the

alkylation reaction with the starting com-

pound The crystal structure analysis of one

selected alkylation product reveals that not

the NN- but the NO-disubstituted deri-

vative was obtained namely ethyl [1-(2-

ethoxy-2-oxoethoxy)-5-methyl-4-oxo-7-

phenylpyrido[34-d]pyridazin-3(4H)-

yl]acetate

References

[1] A Woacutejcicka A Nowicka-Zuchowska Mini-

Rev Org Chem vol 16 3-11 2019 DOI

1021741570193X15666180220155119

[2] A Woacutejcicka L Becan etal III Sympozjum

Szkoła Chemii Medycznej Wroclaw 6-8 września

2017 s[119] pozPP-74

N

N

NH

CH3

O

OH

N

NH

NH

CH3

O

O

(1) (2)


270

Vaping-associated lung injury

Anna Wysoczańska1 Helena Moreira

2 Ewa Barg

2


of Basic Medical Sciences Wroclaw Medical University 2Department of Basic Medical

Sciences Wroclaw Medical University

Vaping is defined as inhaling aerosol pro-

duced by an electronic cigarette E-ciga-

rettes are widely used nowadays which

leads to questions about its health conse-

quences E-liquids consist of propylene

glycol and vegetable glycerin with or

without nicotine and flavouring agents

There are many clinical cases that suggest

vaping can have detrimental impact on the

respiratory system but all the mechanisms

of cellular impairment and possible long-

term effects are still unknown It should be

emphasized that most patients diagnosed

with EVALI (e-cigarette or vaping product

use associated lung injury) admitted using

THC (tetrahydrocannabinol) containing

products which makes the impact of using

e-liquids without the addition of THC even

more uncharted

Clinical cases imply negative impact of

using e-cigarettes on the respiratory system

Frequent symptoms of the vaping-related

lung injury are cough dyspnoea consti-

tutional and gastrointestinal symptoms and

leukocytosis In vitro studies have proved

that vaping causes increased level of free

radicals which leads to oxidative stress and

DNA damage Vaping also results in exces-

sive production of proinflammatory media-

tors such as IL-8 In addition e-liquids

contamination with heavy metals poses

a risk to human health and may cause a lung

injury due to metal accumulation Propylene

glycol and vegetable glycerin (main compo-

nents of e-liquids) after heating to the high

temperature form acetaldehyde formal-

dehyde and acrolein which are considered to

be toxic for human lung bronchial epithelial

cells However all the cytotoxic mecha-

nisms and interactions between e-liquids

compounds are still unknown

In in vitro studies cell cultures exposition to

aerosol which is forming after heating

e-liquid to the proper temperature allows to

evaluate directly the cellular response In

order to understand the mechanism of cyto-

toxicity several research methods are used

including assessment of DNA damage and

apoptosis or the level of intracellular free

radicals and proinflammatory mediators

using lung fibroblast and lung cancer cells

A better understanding of cellular response

to vaping products will provide insight into

the molecular mechanism of EVALI and

will enable development of targeted

treatment

References [1] Chand H Muthumalage T et al Frontiers

in Pharmacology 2020 10 DOI 103389

fphar201901619

[2] Kalininskiy A Bach CT et al The Lancet

Respiratory medicine 2019 7(12) DOI

101016S2213-2600(19)30415-1

[3] Merecz-Sadowska A Sitarek P et al

International Journal of Molecular Sciences 2020

21(2) DOI 103390ijms21020652


271

Influence of nanohydroxyapatite and its compositions on the chosen

oral microorganisms

Wojciech Zakrzewski1 Maciej Dobrzyński

2 Magdalena Pajączkowska

3 Joanna

Nowicka3 Sara Targońska

4 Paulina Sobierajska

4 Katarzyna Wiglusz

5 Maria

Szymonowicz1 Wojciech Dobrzyński1 Adam Lubojański1 Sebastian Fedorowicz3

Zbigniew Rybak1 and Rafal J Wiglusz

4

1Department of Experimental Surgery and Biomaterial Research Wrocław Medical

University Bujwida 44 Wrocław 50-345 Poland 2Department of Conservative Dentistry

and Pedodontics Wrocław Medical University Krakowska 26 50-425 Wrocław Poland 3Department of Microbiology Faculty of Medicine Wrocław Medical University

Chałubińskiego 4 50-368 Wrocław Poland 4Institute of Low Temperature and Structure

Research Polish Academy of Sciences Okoacutelna 2 50-422 Wrocław Poland 5Wrocław

Medical University Faculty of Pharmacy Borowska 211 A 50-566 Wrocław Poland

Background

Nanohydroxyapatite is a material that has

a broad spectrum of use in dentistry It is an

inorganic mineral naturally present in hu-

man hard tissues Copper (Cu) is a neces-

sary trace element in the human body with

antimicrobial activity In case of the ozonated

oil unsaturated fatty acids are released It

should be noted that such products are

commercialized as cosmetic and pharma-

ceutical agents with important charac-

teristics like antibacterial activity This

study is focused on the assessment and

comparison of antimicrobial effects for pure

nanohydroxyapatite copper doped nanohy-

droxyapatite ozonated olive reloaded both

nanohydroxyapatite and copper doped nano-

hydroxyapatite respectively [1]


The measurement were performed using

UVis spectrophotometer electron micros-

copy statistical methods determining the

value of Colony Forming Units (CFUml)

and Minimal Inhibitory Concentration (MIC)

The antimicrobial activity was investigated

against Streptococcus mutans Lactobacillus

rhamnosus and Candida albicans

Results

The pure hexagonal (P63m) apatite phase

was obtained for the materials undoped and

doped with Cu2+ ions The ozonated oil was

successfully nanohydroxyapatite surface

Higher antimicrobial activity was demon-

strated for copper and ozone reloaded

nanohydroxyapatite The applied materials

were effective in the highest sensitivity for

S mutans and the highest resistance for L

rhamnosus


This study was able to create novel biocom-

positions that could find application in

regenerative and esthetic medicine In vitro

screening of microorganism strains accor-

ding to their activity in various experimental

conditions may be a valuable method that

could precede clinical efficacy treatments

References List your references here and use the example

below

[1] Wiglusz RJ Kedziora A et al J Biomed

Nanotechnol vol 8 605-612 2012 DOI

httpsdoiorg 101166jbn20121424


272

The function of transthyretin complexes with metallothionein

in Alzheimerrsquos disease

Natalia Zaręba Marta Kepinska

Department of Biomedical and Environmental Analyses Faculty of Pharmacy Wroclaw

Medical University

Abstract

An important pathological feature of

Alzheimerrsquos disease (AD) is the deposition

of β-amyloid (Aβ) aggregates in extracel-

lular plaques Capture of Aβ protein may

prevent amyloidosis and disorder of this

process can lead to the development of AD

In the context of Aβ plaques formation in

AD there is convincing data suggesting that

one of the factors affecting this mechanism

is the interaction of metallothionein (MT)

with transthyretin (TTR) and the effects of

this interaction depend on which MT

isoform is involved

We report that TTR binds to soluble and

non-aggregated form of Aβ by amino acid

residues on the surface of the monomer

TTR-Aβ interaction results in proteolysis of

Aβ generating smaller peptides and also

degradation of aggregated forms of Aβ

peptide By interaction with TTR MT-2

reduces the ability of TTR to bind to Aβ and

MT-3 causes the opposite effect what

results in the increase of TTR-Aβ binding

The protective effect assigned to MT-3

against deposition of Aβ can just rely on this

mechanism Additionally both Zn7MT-2

and Zn7MT-3 decrease Aβ neurotoxicity in

cultured cortical neurons probably because

of a metal swap between Zn7MT and

Cu(II)Aβ

This report indicates that understanding the

molecular mechanism of metals transfer

between MT and other proteins and still do

not accurately recognized the importance of

the MT isoforms interaction with TTR can

help in AD treatment and prevention

References [1] A L Schwarzman L Gregori et al Proc Natl

Acad Sci U S A vol 91 8368-72 1994

101073pnas91188368

[2] Y Manso PA Adlard et al Metallothionein

and brain inflammation J Biol Inorg Chem vol

16 1103-13 2011 101007s00775-011-0802-y

[3] A Martinho I Gonccedilalves et al FEBS J vol

277 3427-36 2010 101111j1742-46582010

07749x

[4] Z Jiang B Shen et al J Inorg Biochem vol

196 110693 2019 101016jjinorgbio2019

110693

[5] W Xu Q Xu et al SciRep vol 7 13763

2017 101038s41598-017-12800-x

[6] X Ma J Hua et al Inorg Chem vol 57

13533-13543 2018 101021acsinorgchem

8b02115


New pyrrolo[34-d]pyridazinone derivatives as potential

anti-inflammatory and analgesic agents Design synthesis

spectroscopic in vitro and in silico evaluation

A Zborowska1 P Zając

1 K Potyrak

1 K Peregrym

1 B Wiatrak

2 Ł Szczukowski

3

1Student Scientific Club of Chemistry of Drugs Wrocław Medical University Borowska 211

Wrocław Poland 2Department of Basic Medical Sciences Wroclaw Medical University

Borowska 211 Wrocław Poland 3Department of Chemistry of Drugs Wrocław Medical


273

Background Cyclooxygenase a protein which exists in two isoforms ndash constitutive COX-1 and induced COX-2 is strongly involved in inflammation and pain induction Therefore drugs able to inhibit this enzyme are often used as anti-inflammatory and analgesic agents While non-selective cyclooxygenase inhibitors are reported to cause several adverse effects especially gastric irritations selective COX-2 inhibitors known as coxibs cause serious cardiovascular compli-cations [1 2] That is why there is a still urgent prompt to investigate other com-pounds aimed at cyclooxygenase inhibition Pyrrolo[34-d]pyridazinone derivatives have been noticed for their wide range of biological activity Especially anti-inflam-matory and analgesic agents based on this frame are strongly emphasised in recent studies [3 4] Thereby we report herein the design synthesis and biological evaluation of some novel derivatives of this bihete-rocyclic scaffold

Material and Methods In order to reduce gastrointestinal toxicity of new structures a carboxyl group was turned into hydrazide and then final 134-oxiadia-zole-2-thione derivatives were obtained Such modification was inspired by the similar transformations permormed on com-monly used NSAIDs eg Ibuprofenum or Diclofnacum It allows to obtain analgesic and anti-inflammatory compounds with low gastrotoxicity and good affinity towards COX-2 isoform [5] In the last stage of synthesis derivatives of pyrrolo[34-d]py-ridazinone were condensed with appropriate analogues of secondary amine in order to receive final compounds

The structure of novel derivatives was confir-med by spectroscopic methods eg 1H NMR 13C NMR ESI-MS FT-IR and element analysis Cyclooxygenase inhibition activity was examined using in vitro test based on colorimetric evaluation of the

occurrence of the oxidized form of NNNprimeNprime-tetramethyl-p-phenylenediamine (TMPD) which is the substrate for many enzymes with peroxidase activity

Results We determined the concentrations of all examined compounds at which 50 inhi-bition of COX-1 and COX-2 occurred Afterwards IC50 values were calculated Computing the ratio between IC50 values for both cyclooxygenase isoforms allowed us to determine the selectivity of the investigated structures Cellular tests on Normal Human Dermal Fibroblasts (NHDF) were perfor-med in order to evaluate the cytotoxicity of examined derivatives Some in silico stu-dies which estimate potential pharmacolo-gical activity and bioavailability were performed as well

Discussion and conclusions Due to promising biological activity of title compounds some extended pharmacological researches including in vivo tests are plan-ned in near future Such experiments could explain the possible mechanism of action of these derivatives and hopefully confirm their usefulness as effective and safe analge-sics What is more a recent research claim that overexpression of COX-2 is an impor-tant factor in cancer development There-fore our future experiments are also aimed on evaluation of potential chemoprevential activity of new pyrrolo[34-d]pyridazinone derivatives

References [1] K Abouzid et al Bioorganic Med Chem 16 (2008) 5547-5556 [2] Y Boukharsa et al J Mol Struct 1153 (2018) 119-127 [3] W Malinka et al Eur J Med Chem 46 (2011) 4992-4999 [4] S Mogilski et al Pharmacol Biochem Behav 133 (2015) 99-110 [5] MB Palkar et al Bioorganic Med Chem 22 (2014) 2855-2866


274

Spherical nucleic acids ndash characteristic and possible applications

Magdalena Zdrojewska1

1Faculty of Medicine Wroclaw Medical University Poland

Spherical nucleic acids (SNA) are drugs composed of oligonucleotides ndash chains of DNA or RNA encapsulated in liposomal core [1] Scientific reports showed their wide application in the treatment of such diseases as glioblastoma spinal muscular atrophy (SMA) cancer immunotherapy psoriasis In 2016 the American Food and Drug Administration (FDA) approved a drug called Spinraza used in patients with SMA increasing survival and causing far less severe side effects in comparison to the standard SMA drugs [2]

Their spherical shape allows the packing of a large number of nucleic acid molecules into a small volume macromolecule Such dense packing allows for strong interactions with receptors on the surface of the target cells In the cytoplasm of the cells certain sequences of SNAs components act on complementary DNA or RNA sequences of pathogenic molecules [3] The greatest ad-vantage of SNAs is their specificity through the target cells ie there could be designed an oligonucleotide that acts only among cancerous cells with certain mutation [1]

Research shows that pathogenic DNA and RNA binds stronger to SNAs than to free oligonucleotide chains This is due to the fact that genetic material carried by SNAs are densely packed on their core surface [1]

The stiffness of such oligonucleotides is increased which subsequently causes the increase in its torsional tension thus the formation of the bonds with the target is less energy demanding

Some structural features of the SNAs spike ends resemble natural substrates for scavenger receptors ndash responsible for letting the needed nanoparticles into the cell An important feature of SNA is their strong electric charge which protects them from nucleases ndash proteins that destroy foreign DNA and RNA [1 3]

In summary to create SNA it is necessary to produce a large number of oligonucle-otides understand the process of the diseases and develop a way of introducing such oligonucleotides into the appropriate tissues and cells

References [1] H Li et al Molecular spherical nucleic acids Proc Natl Acad Sci U S A vol 115 no 17 pp 4340-4344 Apr 2018 [2] ALTERNATIVE APPROACHES OF CURING SPINAL MUSCLE ATROPHY | Kwartalnik PBK [Online] Available httpspbkomeuplcontent alternative-approaches-curing-spinal-muscle-atrophy [Accessed 16-Feb-2020] [3] Świat Nauki Scientific America Polska Edycja s60-63 wyd22020


The influence of polyphenols on metabolic disorders caused by bisphenol A released from plastics

W Żwierełło 1 M Skoacuterka-Majewicz

1 D Styburski

1 P Kapczuk

2 J Kałduńska

3

J Kikut 3 A Wolska

3 N Komorniak

3 J Palma

3 A Maruszewska

4

1Department of Medical Chemistry Pomeranian Medical University in Szczecin Powst Wlkp 72 Szczecin 70-111 Poland 2Department of Biochemistry and Medical Chemistry Pomeranian Medical University in Szczecin Powst Wlkp 72 Szczecin 70-111 Poland 3Department of Human Nutrition and Metabolomics Pomeranian Medical University in Szczecin Broniewskiego 24 Szczecin 71-460 Poland 4Molecular Biology and Biotechnology Center Institute of Biology University of Szczecin 13 Wąska St Szczecin 71-415 Poland

275

Abstract

Environmental pollution with all kinds of plastics is constantly increasing worldwide The varied and harmful effects of exposure to toxic substances released from plastics inter alia bisphenol A [1 2] contribute to the increase in the incidence of many diseases (eg impairment of liver kidney nervous reproductive and hormonal sys-tems contribute to breast prostate and liver cancers metabolic and neurodegenerative diseases) around the world [3] Continuous exposure of humans and animals to these substances results not only from the slow biodegradation of plastics but also from their ubiquitous use as industrial materials and everyday products [4]

In this paper we discuss the current state of knowledge concerning the protective functions of polyphenols (eg genistein naringenin resveratrol kaempferol curcu-min quercetin silymarin oleuropein hy-droxytyrosol) against the toxic effects of bisphenol A [5-12] We review in detail papers from the last two decades analyzing bisphenol A in terms of their sources of exposure metabolism and toxicity Simul-taneously we demonstrate how polyphenols ndash plant-derived compounds present in a normal daily diet ndash may be used to counteract the harmful environmental effects of such toxins [5-12]

Nutrition has a key impact on the deve-lopment of diseases or protection against those that are associated with exposure to pollution There is increasing evidence of the protective effects of healthy diet on the toxicity of various pollutants particu-larly of a diet rich in polyphenols If con-firmed in clinical trials people could control their risk of developing pollution-related diseases through the right choice of products in their diet or appropriate supplementation [1-4]

To our knowledge this is the first extensive review of in vitro and in vivo studies

concerning the molecular mechanisms of interactions between bisphenol A and polyphenols

References [1] Konieczna A et al Health risk of exposure to Bisphenol A Rocz Panstw Zakl Hig 2015 66 5-11 [2] Konieczna A et al Canned food as a source of bisphenol a (BPA) exposure ndash estimation of consumption among young women from Gdańsk Poland Env Med 2018 21 31-34 [3] Caporossi L Papaleo B Bisphenol A and metabolic diseases Challenges for occupational medicine Int J Environ Res Public Health 2017 14 E959 [4] Rochester J R Bisphenol A and human health A review of the literature Reprod Toxicol 2013 42 132 [5] Li X et al Curcumin modulates miR-19PTENAKTp53 axis to suppress bisphenol A-induced MCF-7 breast cancer cell proliferation Phyther Res 2014c 28 1553-1560 [6] Mahmoudi A et al Oleuropein and hydroxytyrosol protect ratsrsquo pups against bisphenol A induced hypothyroidism Biomed Pharmacother 2018 103 1115-1126 [7] Rameshrad Met al Bisphenol A vascular toxicity protective effect of Vitis vinifera (grape) seed extract and resveratrol Phyther Res 2018 32 2396-2407 [8] Samova S et al The effect of bisphenol A on testicular steroidogenesis and its amelioration by quercetin An in vivo and in silico approach Toxicol Res (Camb) 2018 7 22-31 [9] Wang J et al Cell proliferation and apoptosis in rat mammary glands following combinational exposure to bisphenol A and genistein BMC Cancer 2014 14 379 [10] Zaulet M et al Protective effects of silymarin against bisphenol A-induced hepatotoxicity in mouse liver Exp Ther Med 2017 13 821-828 [11] Bulzomi P et al The naringenin-induced proapoptotic effect in breast cancer cell lines holds out against a high bisphenol a background IUBMB Life 2012 64690-696 [12] Ahangarpour A et al Preventive effects of procyanidin A2 on glucose homeostasis pancreatic and duodenal homebox 1 and glucose transporter 2 gene expression disturbance induced by bisphenol a in male mice J Physiol Pharmacol 2016 67 243-252

276

Index of authors

Adach K 116 218

Andrunioacutew T 222

Anger-Goacutera N 34 37 47 106 153

Anioł M 77 91 197

Antczak O 171

Antoniak M A 60

Augoff K 97

Azarov J E 99

Baczyńska D 77 197

Bagaylyuk L B 258

Balevičiūtė A 59

Banaacuteš D 134

Banaszkiewicz M 52 78 151 180

Banaś K 79 81 181 182 185 186 243

Barg E 166 177 202 270

Bartoszewicz M 100 187

Baurska H 91

Bazylińska U 51 60 246 255

Becan J 268

Belcarz A 45

Benkowska-Biernacka D 82

Berman A 145

Bernikova O G 99

Bielak K 83

Bil-Lula I 52 78 85 122 151 180 208 254 262

Bizoń A 148

Blacha-Grzechnik A 98 142

Błaszczyk M 189 257

Błażkoacutew S 87

Bodnarchuk Yu V 258

Bojanowska-Czajka A 66

Bonarska-Kujawa D 116 171 218 250

Boszkiewcz K 88

Brand I 41

Brożyna M 90 100 187 225

Bryndal I 268

Bryniarski T 145

Brzozowska E 232

Budawsk R 133

Bystranowska D 67 158

Całka J 96 144 191 207

Chajduk E 66

Chmiel K 137

Chmiela M 110 111 112

Choromanska A 234

Chrząstek A 236

Chwiłkowska A 91 201

Chyb M 111 112

Chybicka K 92

Cierluk K 58 93 204

Cierzniak A 94

Cyboran-Mikołajczyk S 171

Cywoniuk P 145

Czajkowska M 96

Czyżnikowska Ż 55 87

Ćwilichowska N 194

Dabrowska A 38

Daczewska M 46 105

Dallolio L 128

Del Giudice A 43

Dembowski J 237

Dobryszycki P 83

Dobrzański T 145

Dobrzyńska D 186

Dobrzyński M 271

Dobrzyński W 271

Dorociak M 97

Dorosz M 66

Duda A 98 142

Durkina A V 99

Dutchak U M 212

Dydak K 90 100 187

Dzianisava V 102 103

Dzida K 226

Eiamphungporn W 248

Elias M 105

Emhemmed F 202

Fecka I 225

Fedorowicz S 271

Ferens-Sieczkowska M 147

Fernandez C 134

Fijałkowski M 116 218

Filik K 232

Fornasier M 43

Franik G 148

Gabrielska J 226

Gajda M 35

Galantini L 43

Gamian A 73

Gdowicz-Kłosok A 165

Gebuza M 123

Geneja M 34 47 106

Gieysztor E 245

Gilowska I 71

Girkontaitė I 59 178

Glatzel-Plucińska N 218

277

Gliński P 208

Gliszczyńska A 205

Gładysz O 210

Gołkowska A 107

Gołyszny M 108

Gomoacutełka M 145

Gonciarz W 110 111 112

Gorczyca N 171

Gorska-Ponikowska M 38

Goszczyński T 37

Goacuterska A 56 113 198 199

Gryziński M 66

Grzesik M 177

Grzeszczak A 115

Grzywacz K 116

Gubernator J 115 170

Gula D 117

Gulla B 214

Guziński M 53

Gwiazdowska A 72

Halecka P 133

Harasym J 79 81 181 182 185 186 243

Hawryłkowicz V 118 263

Heinrich A 119

Hejnowicz M 120

Hładyszowski J 220

Hof M 226

Hołub S 121

Hosnedlovaacute B 134

Ivantsiv O R 258

Iwankiewicz J 91

Jabłońska J 63 69 192 247

Jabłońśki S 174

Jacewicz D 38

Jachimska B 41 167 233

Jana B 96 144 191 207

Janik M 122

Janiszewska E 71

Jankowska A M 93

Jarocki M 123

Jędryka M 139 219

Jędrzejak M 124

Junka A 90 100 187

Jurga A 194

Jurkowska K 237

Kabaj M 125 136 229 241

Kaczorowska A 92

Kalinowski L 38

Kaliszewski K 94

Kałas W 92

Kałduńska J 126 133 140 141 190 215

228 274

Kanclerz G 48

Kapczuk P 127 190 215 228 274

Karolewicz B 35 107

Kawalec A 128 130

Kazimierczak W 46

Kepinska M 33 40 117 134 159 272

Kęsik M 141

Khan N A 49 131

Khan T A 28 49 131

Kichler K 232

Kicia M 235

Kielan W 53

Kiełbasa E 117

Kiełbik A 58 132 204

Kikut J 126 133 140 141 190 215 228 274

Kizek R 26 134 223 248 252

Klak M 145

Klimas K 125 136 229 241

Kłosiński T 137

Kmieć Z 38

Kokot I 71 139

Komorek P 41

Komorniak N 118 126 127 133 140 141

190 215 228 263 274

Konecka N 126 133 140 141 190 215 228

Kopyciński B 98 142

Kordylewska I 144

Kosowska K 145

Kotas M 250

Kotowska A 155

Kotyra Ł 225

Kowalska P 145

Kowalski K 146

Kozakiewicz M 35

Kozioł A 147

Kozłowska J 77 197

Kral T 226

Krasoń M 148

Kraszkiewicz W 149

Kratz E M 71 139 219

Krawczyk M S 150

Krotkiewski H 219

Kroacutel M 33

Krupa A 110

Krześniak M 165

Krzywonos-Zawadzka A 52 78 85 151 180

Kuban-Jankowska A 38

Kulbacka J 46 51 53 56 58 106 121

278

132 146 153 169 172 198 199 204 256

Kurzyna W 154

Kuzan A 155

Kuźma J 194

Kwiecień A 156

Lachowicz S 171

Lenda R 158

Leszek J 64

Lewandowicz-Uszyńska A 73

Lewandowski Ł 33 40 159

Lewoń D 161

Ligenza A 154

Lipke K 162

Lochyński S 72

Lubojański A 271

Ludyga T 108

Lupa D 163

Łabowska M B 93

Łasut-Szyszka B 165

Łukaszewicz M 174

Machowska M 102 103

Machynia M 166

Madej P 148

Magnus Rae J 167

Majerz I 150 156 238 239

Majgier K 169

Majumdar S 131

Malyško V 59

Małodobra-Mazur M 94

Małolepsza-Jarmołowska K 216

Małysa A 179

Markowski A 170

Maruszewska A 274

Matczyszyn K 62 82

Matera-Witkiewicz A 175 265

Matoszka M 240

Matuszczyk I 165

Matuszewska A 119

Matuszewska K 166

Matuszewski M 237

Matyśkiewicz P 154

Mazur B 214

Mazurek W 120

Menezes S 49

Męczarska K 116 171

Michalak K 147 189 257

Michalicha A 45

Michel O 58 132 172

Mielko K A 174

Mierzejewska J 34 37 47 106

Migocka-Patrzałek M 105

Mikołajczyk A 175 265

Milczanowska A 154

Milnerowicz H 33 40 134 159 161 183

Miskiv V A 258

Młynarz P 174

Molnar J 257

Momin M 49 131

Mooney J 233

Moreira H 166 177 202 270

Mosser M 85

Mucha I 260

Muller C D 202

Murauska A 178

Murgia S 43

Naporowski P 73

Nartowski K P 35 107

Nawaryński M 260

Nawrot K C 51

Niedbalska-Tarnowska J 105

Niezgoda N 205

Novickij J 59

Novickij V 59 178

Nowak K 48

Nowak S 62

Nowakowska K 155

Nowicka J 271

Nowiczuk E 124

Nussberger S 38

Nyk M 51 60

Obuchowicz E 108

Ochnik M 64

Olbromski M 218

Olchowy A 179

Olchowy C 179

Olejnik A 52 78 151 180

Olewniak-Adamowska A 38

Olędzki R 79 81 181 182 185 186 243

Ołdakowska M 183

Orkusz A 79 81 181 182 185 186 243

Ożyhar A 67

Pajączkowska M 271

Pajtasz-Piasecka E 34 37 47

Pakhomova O 29

Paleczny J 90 100 187

Palko-Łabuz A 147 189

Palma J 126 133 140 141 190 215 228 274

Palus K 191

Pałka M 63 69 192 247

Patel K 131

279

Paul-Samojedny M 108

Pawlak A 92

Pawlas K 128 130

Pawlik K 62

Pecuch A 245

Peregrym K 272

Piasecki E 64

Piekarowicz K102 103

Piksa M 62

Pireddu R 43

Piscitelli G 128

Piszko P 194

Piwowar A 88 139 162 236 237

Ploska A 38

Płaczkowska S 122

Płochocki M 53

Pormańczuk K 208

Potyrak K 272

Pruchnik H 195 267

Przybyła N 141

Przybyszewski O 197 202

Przychodzen P 38

Przystupski D 56 113 198 199

Pyra A 268

Pytel J 201

Radajewska A 202

Rak-Pasikowska A 208 262

Raszewski Z 203

Redzicka A 137

Rembiałkowska N 121 153 204

Rogalski K 87

Rojek J 82

Rossowska J 34 37 47 106 153

Rudno-Rudzińska J 53

Rusin M 165

Rutkowska M 119

Ruttkay-Nedeckyacute B 134 223 248

Růžička J 223 248

Rybak Z 271

Rychlicka M 205

Rząp D 207

Rzepecki R 63 69 102 103 192 247

Saczko J 58 77 132 172 204

Sapa-Wojciechowska A 208

Sarna M 133

Sarzyński D 210

Sawicka E 88 154 225 231 237

Sawicki G 85

Schilleacuten K 43

Sedova K A 99

Sehnal K 134

Seweryn P 179

Shah S 131

Shchur M B 211

Siadak A 195

Sinico C 43

Skibiński P 210

Skora J S 211 212

Skorupska A 67

Skorupski M 214

Skoacuterka M 127 141

Skoacuterka-Majewicz M 126 133 140 190

215 228 274

Smardz J 179

Smoleński M 216

Sobczyński M 64

Sobierajska P 271

Sochocka M 64

Solarska-Ściuk K 116 218

Sołkiewicz K 219

Sonetha V 131

Spiegel M 220 222

Spinek A 250

Sroka A 156

Sroka Z 220 222

Staigvila G 178

Staňkovaacute M 134 223 248 252

Starzec A 90 225

Starzyńska A 225

Stasiak S 225

Staszuk M 142

Stebnicka A 155

Stolarczyk M 175 265

Strub D J 72

Strugała P 226

Styburski D 126 127 133 140 141 190

215 228 274

Supplit S 58

Surowiak A K 72

Sycz Z 125 136 229 235 241

Szafraniec M 231

Szczepańska P 231

Szczukowski Ł 272

Szczygieł A 34 37 47 106

Szermer-Olearnik B 47 232

Szewczyk A 46 56 58 204

Szewczyk M 133

Szlasa W 58 132 172 204

Szota M 233

Szwedowicz U 234

280

Szydełko M 185

Szydłowicz M 229 235

Szyjka A 177

Szymańska B 236 237

Szymańska M 239

Szymański S 238

Szymonowicz M 271

Ściskalska M 161 183

Śliwińska-Hill U 240

Śliwińska-Mossoń M 254

Środa-Pomianek K 147 189 257

Święcicka-Klama A 125 136 229 241

Świtalska M 37

Tancula N 79 81 181 182 185 186 243

Tarek M 25 58 172 204

Targońska S 271

Telenga M 245

Tokarek B 246

Tomaszewska A 111 112

Tomczak A 63 69 192 247

Toacutethovaacute Z 223 248 252

Trukan A 225

Tunikowska J 27

Turek K 123

Turowski P 145

Tyliszczak M 250

Tymicki G 145

Tyszer J 250

Uhliacuteřovaacute D 134 223 248 252

Urbanowicz I 33 40 159

Uryga A 189

Vasyliuk V M 212

Vlasiuk T I 212

Všetičkovaacute M 134 223 248 252

Wadowska K 254

Waglewska E 255

Wala K 256

Wanarska E 42

Wesołowska O 147 189 257

Weżgowiec J 179

Węgierek K 34 37 47 106

Wiatr M 258

Wiatrak B 177 272

Więckiewicz M 179

Wiglusz J 271

Wiglusz K 260 271

Wiglusz R J 260

Wiśniewska K 262

Witkowska D 73 231

Włoch A 195 267

WłochA 116

Włodarczyk M 112

Wojcieszonek A 118 263

Wojnicz D 229

Wolańska E 245

Wolska A 175 265 274

Worobiec M 267

Woźniak M 122

Woacutejcicka A 268

Woacutejcik M 69

Woacutejciuk G 66

Woacutejciuk K 66

Wrona A 149

Wroacuteblewska A 232

Wrzyszcz A 262

Wszoła M 145

Wysoczańska A 270

Zając P 272

Zajączkowska Ż 235

Zajkowicz A 165

Zakrzewski W 271

Zalewski J 48

Zaremba-Czogalla M 115 170

Zaręba N 272

Zborowska A 272

Zdrojewska M 274

Želvys A 59

Zhurakivska O V 212

Zhurakivska O Ya 212

Zhurakivskyi V M 258

Zieliński M 108

Zinkevičienė A 59 178

Zioło E 92

Złocińska A 35

Zmijewski M A 38

Zoglowek A 83

Zwolińska K 64

Zygmunt A 115

Żak A 62

Żak M 149

Żbikowska B 220

Żwierełło W 126 127 133 140 190 215

228 274

4



Editors

Julita Kulbacka

Nina Rembiałkowska

Joanna Weżgowiec

Wroclaw 2020

4th


Editors

Julita Kulbacka

Nina Rembiałkowska

Joanna Weżgowiec

Typesetting

Monika Maciąg

Kamil Maciąg

Cover design

Marcin Szklarczyk

Nina Rembiałkowska


ISBN 978-83-66489-37-0

Publisher




Scientific Committe








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Poland




Poland


















Organizing Committe




Poland





Poland









Poland
















Organizers

Honorary Patronage





Sponsors


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NIP 118-178-86-00

tel22722-70-48 do 50

fax 22722-70-51

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Content


LECTURES 23




CONFERENCE PROGRAM

19

09102020 Friday


1st SESSION


2nd



20

3rd




in dogs and cats






Kanclerz Gabriela


Menezes










10102020 Saturday

4

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Malyško Veronika





21

5th

SESSION


6th




7th




8th





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25

L 1



chemistry

Mounir Tarek


FRANCE












26

L 2


Rene Kizek


































27

L 3


and cats

Joanna Tunikowska
























5616(02)00002-5






28

L 4



Tabassum Asif Khan






























29

L 5


Olga Pakhomova


USA















ORAL PRESENTATIONS

33

Session 1 O 1





Urbanowicz


3 Marta Kepinska

3

Halina Milnerowicz3





Background


































kits

Results




















IL-6 production [4]








34





obesity and T2D



101007s10787-018-0458-0




2017019712


58 11 2009 DOI 101007s00011-009-0060-4


99 16 1999



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1 Magdalena Geneja

1 Natalia


1 Joanna Rossowska


1







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Session 1 O 3



printing technology




1

Karol P Nartowski1




Background


















methods


36

Results













tablets with SIM







presented [2]





























Sci 2018 doi101016jtips 201802006




201470(1)81-90doi101107S2052520613026917



37

Session 1 O 4






1 Jagoda


1 Marta Świtalska

1 Joanna Rossowska

1








38

Results


















growth












growth



and 201727BNZ602702)


2014 e00047


37 126 doi 101186s13046-018-0799-y


Session 1 O 5





2


3 Zbigniew Kmieć

2 Agata Ploska

45


6 Leszek Kalinowski

46 Magdalena






Background













39


























scopy techniques

Results













cell death




















One13(12)e0209489 2018 doi 101371journal

pone0209489


15(4)297-310 2006 DOI101111j1750-

36392005tb00115x



116303356


1109-34 2016 doi 101016jsteroids201603017


Pharmacol 68(2)293-300 2011 doi

101007s00280-010-1478-7



oncotarget3913



101016jbiopha201812051


Genet 28(2)177-99 2019 doi 101093hmg

ddy335


40

Session 1 O 6




2 Marta Kepinska


1




Background














of SODs
















group respectively















literature









ELISA kit)

Results
















41








Session 2 O 1


the gold surface


2 Barbara Jachimska

1




42








Session 2 O 2


nanoparticles

Ewelina Wanarska1


and Technology

43

Background




Results






cells












References


Diseases vol 48 1-12 2009 101086595011



103389fmicb201701002


vol 33 2015 103389fmicb201701002



12 215-220 2015 101016jpdpdt201503003


Session 2 O 3



M Fornasier12


3 A Del Giudice

3 C Sinico

4 R Pireddu

4

S Murgia1




of Cagliari

44

Background





















antioxidants












Results







Cryo-Tem (Fig 1)



































27 13461-13467 2011 DOI number 101021la

203028s



1999 DOI number 101006jcis19985996







45



Session 2 O 4



1


Background










and Zhang 2017)












































46



















weight increase

Results





































against infection


68 128-135 2017 101016jfoodhyd201609014






Session 2 O 5





1 Julita

Kulbacka2





47



































zebrafish model

References



101186s13000-016-0552-9






Session 3 O 1






1 Agnieszka



1 Joanna Rossowska

1




Background




















48

























Results
























NZ402834 201727BNZ602702)


449419-26 2007


12 265-277 2012


Session 3 O 2


ndash survey study


1 Jaroslaw Zalewski

2




Background








students




49
































Results




































378-83 doi 101016jpec201411019


Session 3 O 3



1 Tabassum Khan

2 and Munira Momin

2



50








51



Session 3 O 4


bioimaging


2 Julita Kulbacka

3 Marcin Nyk

1








52




Acknowledgements



Session 3 O 5





1 Iwona

Bil-Lula1







53









Session 3 O 6




2 Maciej Guziński

3 Maciej Płochocki

4

Wojciech Kielan1


54












55

Session 4 O 1





Background












































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Results














Val233 residues

















56







studies

Acknowledgments






101002med21436



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Chemistry vol 18 1151-1157 2018 102174

1389557516666160620095103



101016jejmech201806041



Session 4 O 2



2 Anna Szewczyk

2 Julita Kulbacka

2



Background
















Machine (RPM)






57


















microscope

Results

















Conclusions


















for 2h on RPM


58

Session 4 O 3



2 Karolina Cierluk


1 Stanisław


4 Anna Szewczyk

25 Jolanta Saczko

2 Julita Kulbacka

2






Background













beneficial [2]


















Results














much worse results

















59

Acknowledgments









support




1864 2019






pp 145-152 Feb 2015


Session 4 O 4


Rate




2



2


Vilnius Lithuania

Background






































pulse delivery







60


(Amnis Seattle USA)






Results



























rements



therapy

Conclusion






Acknowledgement



22












Session 4 O 5



Magda A Antoniak1


1





61







References




62

Session 4 O 6



Andrzej Żak1 2


2 Sylwia Nowak

3 Katarzyna

Matczyszyn4





Background








effect of aPDT


















Results











bacterial death












nanometric scale




XNZ302100



101111php12733


101038s41528-019-0058-0

63



Session 6 O 1





1 Ryszard Rzepecki

1


Background




































occurred during HSR








64
















cRAP)






Session 6 O 2


Marta Sochocka1 and

Michał Ochnik



1


3


65

Background













































Results





























viruses













to a minimum

66



101007s12223-016-0482-7


854-61 2014 DOI 101016jcoviro201406003


28 746-752 2014 DOI 10108014786419

2013879303




423496 18-11-2017)


Session 6 O 3



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Results


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Limited proteolysis








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Inhibition

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A - 36 765 nM

B - 35 821 nM

C - 31 4 M

D - 31 4 M

E - 31 3 M

F - 35 843 nM









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99-108 2007


2010


30(11)e13439 2018 doi 101111nmo13439






1 Ryszard Rzepecki

1


Background













occurs [1]


193









194



2 Joanna Kuźma

3 Anna Jurga

4















195






and cell membranes


2 Aleksandra Włoch

1




Background


















196














of RhTCEP






































cells





















-) of











Conclusions






197




Chichester 2005












2 Mirosław Anioł


3





Poland

Background





























Results













gene expression






References

198










101002jcb25729






24040679





2 Julita Kulbacka

2



Background

























































vol 72 pp 49-64 Jun 2014

199






campaign


1 Julita Kulbacka

1


Background
















200



















































201





2








202






1 Helena Moreira

2 Ewa Barg

2


3









203


Zbigniew Raszewski














204








2 Karolina Cierluk


2


4 Jolanta Saczko

1 Julita Kulbacka

1




F-54000 Nancy

Background














205



























Results

























Aug 2013





SA 2019





University





Poland

Background










206



























42 minutes [5]










body






































207










2 Jarosław Całka

1







208










1 Paweł Gliński

2 Kornel

Pormańczuk34

Iwona Bil-Lula1




209

















reference tissue










Results






in Table 1




Total

cholesterol

r = 02791


r = 009936

p = 06768

LDL-

cholesterol

r = 0225

p = 03403

White blood

count

r = -03164

p = 01741

HDL-

cholesterol

r = 01734

p = 04647

Platelet

count

r = 03544

p = 01252

Non-HDL-

cholesterol

r = 02143


r = -01964

p = 04066



















required [2 4]

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H3

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H3

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H3

OH OC

H3

H

4-

hydorxybenz

oic

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H OH H H H

protocatechui

c

COOH H OH OH H H

vanillic COOH H OC

H3

OH H H


3-

hydroxybenz

oic

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o-coumaric CH=CHC

OOH

O

H

H H H H

23-

dihydroxyben

COOH O

H

OH H H H

zoic

salicylic COOH O

H

H H H H

sinapic CH=CHC

OOH

H OC

H3

OH OC

H3

H

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H3

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H3

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241












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242









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12 Karol Banaś

1 Agnieszka Orkusz

12 Remigiusz

Olędzki12

















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12

Agnieszka Orkusz12




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baking ingredient














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nutrients




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1 Ewa Gieysztor

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also defined

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1 Ryszard Rzepecki

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248











of zinc (II) ions




1


3 Josef Růžička

1 Reneacute

Kizek12




249









250



1













3


251

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Results





and figure 2





















of material


1328-1331 1965










252









1

Rene Kizek12




Background









0

10

20

30

40

50

370 390 410 430 450 470 490 510 530 550

Inte

nsi

ty [

au]

Wavelengt [nm]


Groszowice 8


Milicz 80




0

10

20

30

40

50

380 400 420 440 460 480 500 520 540 560

Inte

nsi

ty [

au]

Wavelengt [nm]


Groszowice 8


Milicz 80




253




















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of 625 313 156 78 39 and 0 was













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Wroclaw Poland









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256











































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nervous system [1]






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258





























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Results









































127 2008 DOI 1022171462241691105





2 Zhurakivskyi VM

3 Miskiv VA

4 Bodnarchuk YuV

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Background

















chronic stress




















bioethics commitee

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DM



























260




















































2019








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Background










261





















Results

















drugs











binding




899-901


262




1 Aneta Wrzyszcz

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CD63

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263























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265






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platinum (IC50 = 6368) [1]













tested before






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Results












268
































ndash PdTCEP [1]





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1 Iwona Bryndal

1



269

Background




















Results






























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2017 s[119] pozPP-74

N

N

NH

CH3

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OH

N

NH

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CH3

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272





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isoform is involved

















Cu(II)Aβ








Acad Sci U S A vol 91 8368-72 1994

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274














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275

Abstract







276

Index of authors

Adach K 116 218



Anioł M 77 91 197

Antczak O 171

Antoniak M A 60

Augoff K 97

Azarov J E 99

Baczyńska D 77 197

Bagaylyuk L B 258

Balevičiūtė A 59

Banaacuteš D 134


Banaś K 79 81 181 182 185 186 243

Barg E 166 177 202 270


Baurska H 91


Becan J 268

Belcarz A 45


Berman A 145

Bernikova O G 99

Bielak K 83

Bil-Lula I 52 78 85 122 151 180 208 254 262

Bizoń A 148



Błażkoacutew S 87

Bodnarchuk Yu V 258



Boszkiewcz K 88

Brand I 41

Brożyna M 90 100 187 225

Bryndal I 268

Bryniarski T 145

Brzozowska E 232

Budawsk R 133


Całka J 96 144 191 207

Chajduk E 66

Chmiel K 137

Chmiela M 110 111 112

Choromanska A 234

Chrząstek A 236


Chyb M 111 112

Chybicka K 92

Cierluk K 58 93 204

Cierzniak A 94


Cywoniuk P 145

Czajkowska M 96


Ćwilichowska N 194

Dabrowska A 38

Daczewska M 46 105

Dallolio L 128

Del Giudice A 43

Dembowski J 237

Dobryszycki P 83

Dobrzański T 145

Dobrzyńska D 186

Dobrzyński M 271

Dobrzyński W 271

Dorociak M 97

Dorosz M 66

Duda A 98 142

Durkina A V 99

Dutchak U M 212

Dydak K 90 100 187


Dzida K 226

Eiamphungporn W 248

Elias M 105

Emhemmed F 202

Fecka I 225

Fedorowicz S 271


Fernandez C 134


Filik K 232

Fornasier M 43

Franik G 148

Gabrielska J 226

Gajda M 35

Galantini L 43

Gamian A 73


Gebuza M 123

Geneja M 34 47 106

Gieysztor E 245

Gilowska I 71



277

Gliński P 208

Gliszczyńska A 205

Gładysz O 210

Gołkowska A 107

Gołyszny M 108

Gomoacutełka M 145


Gorczyca N 171


Goszczyński T 37


Gryziński M 66

Grzesik M 177

Grzeszczak A 115

Grzywacz K 116


Gula D 117

Gulla B 214

Guziński M 53

Gwiazdowska A 72

Halecka P 133

Harasym J 79 81 181 182 185 186 243


Heinrich A 119

Hejnowicz M 120

Hładyszowski J 220

Hof M 226

Hołub S 121


Ivantsiv O R 258

Iwankiewicz J 91


Jabłońśki S 174

Jacewicz D 38


Jana B 96 144 191 207

Janik M 122

Janiszewska E 71

Jankowska A M 93

Jarocki M 123

Jędryka M 139 219

Jędrzejak M 124

Junka A 90 100 187

Jurga A 194

Jurkowska K 237

Kabaj M 125 136 229 241

Kaczorowska A 92

Kalinowski L 38

Kaliszewski K 94

Kałas W 92

Kałduńska J 126 133 140 141 190 215

228 274

Kanclerz G 48

Kapczuk P 127 190 215 228 274

Karolewicz B 35 107

Kawalec A 128 130

Kazimierczak W 46

Kepinska M 33 40 117 134 159 272

Kęsik M 141

Khan N A 49 131

Khan T A 28 49 131

Kichler K 232

Kicia M 235

Kielan W 53

Kiełbasa E 117


Kikut J 126 133 140 141 190 215 228 274

Kizek R 26 134 223 248 252

Klak M 145

Klimas K 125 136 229 241

Kłosiński T 137

Kmieć Z 38

Kokot I 71 139

Komorek P 41

Komorniak N 118 126 127 133 140 141

190 215 228 263 274

Konecka N 126 133 140 141 190 215 228


Kordylewska I 144

Kosowska K 145

Kotas M 250

Kotowska A 155

Kotyra Ł 225

Kowalska P 145

Kowalski K 146

Kozakiewicz M 35

Kozioł A 147

Kozłowska J 77 197

Kral T 226

Krasoń M 148

Kraszkiewicz W 149

Kratz E M 71 139 219

Krawczyk M S 150

Krotkiewski H 219

Kroacutel M 33

Krupa A 110

Krześniak M 165



Kulbacka J 46 51 53 56 58 106 121

278

132 146 153 169 172 198 199 204 256

Kurzyna W 154

Kuzan A 155

Kuźma J 194

Kwiecień A 156

Lachowicz S 171

Lenda R 158

Leszek J 64



Lewoń D 161

Ligenza A 154

Lipke K 162

Lochyński S 72

Lubojański A 271

Ludyga T 108

Lupa D 163

Łabowska M B 93


Łukaszewicz M 174

Machowska M 102 103

Machynia M 166

Madej P 148

Magnus Rae J 167

Majerz I 150 156 238 239

Majgier K 169

Majumdar S 131

Malyško V 59



Małysa A 179

Markowski A 170

Maruszewska A 274

Matczyszyn K 62 82


Matoszka M 240

Matuszczyk I 165

Matuszewska A 119

Matuszewska K 166

Matuszewski M 237

Matyśkiewicz P 154

Mazur B 214

Mazurek W 120

Menezes S 49



Michalicha A 45

Michel O 58 132 172

Mielko K A 174




Milczanowska A 154

Milnerowicz H 33 40 134 159 161 183

Miskiv V A 258

Młynarz P 174

Molnar J 257

Momin M 49 131

Mooney J 233

Moreira H 166 177 202 270

Mosser M 85

Mucha I 260

Muller C D 202

Murauska A 178

Murgia S 43

Naporowski P 73


Nawaryński M 260

Nawrot K C 51


Niezgoda N 205

Novickij J 59

Novickij V 59 178

Nowak K 48

Nowak S 62

Nowakowska K 155

Nowicka J 271

Nowiczuk E 124

Nussberger S 38

Nyk M 51 60

Obuchowicz E 108

Ochnik M 64

Olbromski M 218

Olchowy A 179

Olchowy C 179

Olejnik A 52 78 151 180


Olędzki R 79 81 181 182 185 186 243

Ołdakowska M 183

Orkusz A 79 81 181 182 185 186 243

Ożyhar A 67

Pajączkowska M 271


Pakhomova O 29



Palma J 126 133 140 141 190 215 228 274

Palus K 191

Pałka M 63 69 192 247

Patel K 131

279


Pawlak A 92

Pawlas K 128 130

Pawlik K 62

Pecuch A 245

Peregrym K 272

Piasecki E 64


Piksa M 62

Pireddu R 43

Piscitelli G 128

Piszko P 194

Piwowar A 88 139 162 236 237

Ploska A 38

Płaczkowska S 122

Płochocki M 53

Pormańczuk K 208

Potyrak K 272

Pruchnik H 195 267

Przybyła N 141


Przychodzen P 38


Pyra A 268

Pytel J 201

Radajewska A 202


Raszewski Z 203

Redzicka A 137


Rogalski K 87

Rojek J 82

Rossowska J 34 37 47 106 153


Rusin M 165

Rutkowska M 119



Rybak Z 271

Rychlicka M 205

Rząp D 207

Rzepecki R 63 69 102 103 192 247

Saczko J 58 77 132 172 204


Sarna M 133

Sarzyński D 210

Sawicka E 88 154 225 231 237

Sawicki G 85

Schilleacuten K 43

Sedova K A 99

Sehnal K 134

Seweryn P 179

Shah S 131

Shchur M B 211

Siadak A 195

Sinico C 43

Skibiński P 210

Skora J S 211 212

Skorupska A 67

Skorupski M 214



215 228 274

Smardz J 179

Smoleński M 216

Sobczyński M 64

Sobierajska P 271

Sochocka M 64


Sołkiewicz K 219

Sonetha V 131

Spiegel M 220 222

Spinek A 250

Sroka A 156

Sroka Z 220 222

Staigvila G 178


Starzec A 90 225

Starzyńska A 225

Stasiak S 225

Staszuk M 142

Stebnicka A 155


Strub D J 72

Strugała P 226

Styburski D 126 127 133 140 141 190

215 228 274

Supplit S 58

Surowiak A K 72

Sycz Z 125 136 229 235 241

Szafraniec M 231

Szczepańska P 231

Szczukowski Ł 272



Szewczyk A 46 56 58 204

Szewczyk M 133

Szlasa W 58 132 172 204

Szota M 233

Szwedowicz U 234

280

Szydełko M 185


Szyjka A 177


Szymańska M 239

Szymański S 238

Szymonowicz M 271






Świtalska M 37

Tancula N 79 81 181 182 185 186 243

Tarek M 25 58 172 204

Targońska S 271

Telenga M 245

Tokarek B 246


Tomczak A 63 69 192 247


Trukan A 225

Tunikowska J 27

Turek K 123

Turowski P 145

Tyliszczak M 250

Tymicki G 145

Tyszer J 250



Uryga A 189

Vasyliuk V M 212

Vlasiuk T I 212


Wadowska K 254

Waglewska E 255

Wala K 256

Wanarska E 42


Weżgowiec J 179

Węgierek K 34 37 47 106

Wiatr M 258

Wiatrak B 177 272

Więckiewicz M 179

Wiglusz J 271

Wiglusz K 260 271

Wiglusz R J 260

Wiśniewska K 262

Witkowska D 73 231

Włoch A 195 267

WłochA 116

Włodarczyk M 112


Wojnicz D 229

Wolańska E 245

Wolska A 175 265 274

Worobiec M 267

Woźniak M 122

Woacutejcicka A 268

Woacutejcik M 69

Woacutejciuk G 66

Woacutejciuk K 66

Wrona A 149


Wrzyszcz A 262

Wszoła M 145

Wysoczańska A 270

Zając P 272


Zajkowicz A 165

Zakrzewski W 271

Zalewski J 48


Zaręba N 272

Zborowska A 272

Zdrojewska M 274

Želvys A 59

Zhurakivska O V 212



Zieliński M 108


Zioło E 92

Złocińska A 35

Zmijewski M A 38

Zoglowek A 83

Zwolińska K 64

Zygmunt A 115

Żak A 62

Żak M 149

Żbikowska B 220

Żwierełło W 126 127 133 140 190 215

228 274

4th


Editors

Julita Kulbacka

Nina Rembiałkowska

Joanna Weżgowiec

Typesetting

Monika Maciąg

Kamil Maciąg

Cover design

Marcin Szklarczyk

Nina Rembiałkowska


ISBN 978-83-66489-37-0

Publisher




Scientific Committe








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Content


LECTURES 23




CONFERENCE PROGRAM

19

09102020 Friday


1st SESSION


2nd



20

3rd




in dogs and cats






Kanclerz Gabriela


Menezes










10102020 Saturday

4

th SESSION








Malyško Veronika





21

5th

SESSION


6th




7th




8th





LECTURES

25

L 1



chemistry

Mounir Tarek


FRANCE












26

L 2


Rene Kizek


































27

L 3


and cats

Joanna Tunikowska
























5616(02)00002-5






28

L 4



Tabassum Asif Khan






























29

L 5


Olga Pakhomova


USA















ORAL PRESENTATIONS

33

Session 1 O 1





Urbanowicz


3 Marta Kepinska

3

Halina Milnerowicz3





Background


































kits

Results




















IL-6 production [4]








34





obesity and T2D



101007s10787-018-0458-0




2017019712


58 11 2009 DOI 101007s00011-009-0060-4


99 16 1999



1011552013976810


Session 1 O 2




1 Magdalena Geneja

1 Natalia


1 Joanna Rossowska


1







35






Session 1 O 3



printing technology




1

Karol P Nartowski1




Background


















methods


36

Results













tablets with SIM







presented [2]





























Sci 2018 doi101016jtips 201802006




201470(1)81-90doi101107S2052520613026917



37

Session 1 O 4






1 Jagoda


1 Marta Świtalska

1 Joanna Rossowska

1








38

Results


















growth












growth



and 201727BNZ602702)


2014 e00047


37 126 doi 101186s13046-018-0799-y


Session 1 O 5





2


3 Zbigniew Kmieć

2 Agata Ploska

45


6 Leszek Kalinowski

46 Magdalena






Background













39


























scopy techniques

Results













cell death




















One13(12)e0209489 2018 doi 101371journal

pone0209489


15(4)297-310 2006 DOI101111j1750-

36392005tb00115x



116303356


1109-34 2016 doi 101016jsteroids201603017


Pharmacol 68(2)293-300 2011 doi

101007s00280-010-1478-7



oncotarget3913



101016jbiopha201812051


Genet 28(2)177-99 2019 doi 101093hmg

ddy335


40

Session 1 O 6




2 Marta Kepinska


1




Background














of SODs
















group respectively















literature









ELISA kit)

Results
















41








Session 2 O 1


the gold surface


2 Barbara Jachimska

1




42








Session 2 O 2


nanoparticles

Ewelina Wanarska1


and Technology

43

Background




Results






cells












References


Diseases vol 48 1-12 2009 101086595011



103389fmicb201701002


vol 33 2015 103389fmicb201701002



12 215-220 2015 101016jpdpdt201503003


Session 2 O 3



M Fornasier12


3 A Del Giudice

3 C Sinico

4 R Pireddu

4

S Murgia1




of Cagliari

44

Background





















antioxidants












Results







Cryo-Tem (Fig 1)



































27 13461-13467 2011 DOI number 101021la

203028s



1999 DOI number 101006jcis19985996







45



Session 2 O 4



1


Background










and Zhang 2017)












































46



















weight increase

Results





































against infection


68 128-135 2017 101016jfoodhyd201609014






Session 2 O 5





1 Julita

Kulbacka2





47



































zebrafish model

References



101186s13000-016-0552-9






Session 3 O 1






1 Agnieszka



1 Joanna Rossowska

1




Background




















48

























Results
























NZ402834 201727BNZ602702)


449419-26 2007


12 265-277 2012


Session 3 O 2


ndash survey study


1 Jaroslaw Zalewski

2




Background








students




49
































Results




































378-83 doi 101016jpec201411019


Session 3 O 3



1 Tabassum Khan

2 and Munira Momin

2



50








51



Session 3 O 4


bioimaging


2 Julita Kulbacka

3 Marcin Nyk

1








52




Acknowledgements



Session 3 O 5





1 Iwona

Bil-Lula1







53









Session 3 O 6




2 Maciej Guziński

3 Maciej Płochocki

4

Wojciech Kielan1


54












55

Session 4 O 1





Background












































was performed

Results














Val233 residues

















56







studies

Acknowledgments






101002med21436



101016jejmech201810028


Chemistry vol 18 1151-1157 2018 102174

1389557516666160620095103



101016jejmech201806041



Session 4 O 2



2 Anna Szewczyk

2 Julita Kulbacka

2



Background
















Machine (RPM)






57


















microscope

Results

















Conclusions


















for 2h on RPM


58

Session 4 O 3



2 Karolina Cierluk


1 Stanisław


4 Anna Szewczyk

25 Jolanta Saczko

2 Julita Kulbacka

2






Background













beneficial [2]


















Results














much worse results

















59

Acknowledgments









support




1864 2019






pp 145-152 Feb 2015


Session 4 O 4


Rate




2



2


Vilnius Lithuania

Background






































pulse delivery







60


(Amnis Seattle USA)






Results



























rements



therapy

Conclusion






Acknowledgement



22












Session 4 O 5



Magda A Antoniak1


1





61







References




62

Session 4 O 6



Andrzej Żak1 2


2 Sylwia Nowak

3 Katarzyna

Matczyszyn4





Background








effect of aPDT


















Results











bacterial death












nanometric scale




XNZ302100



101111php12733


101038s41528-019-0058-0

63



Session 6 O 1





1 Ryszard Rzepecki

1


Background




































occurred during HSR








64
















cRAP)






Session 6 O 2


Marta Sochocka1 and

Michał Ochnik



1


3


65

Background













































Results





























viruses













to a minimum

66



101007s12223-016-0482-7


854-61 2014 DOI 101016jcoviro201406003


28 746-752 2014 DOI 10108014786419

2013879303




423496 18-11-2017)


Session 6 O 3



2 Ewelina Chajduk

2 Michał Dorosz

1


4


Background




















































67


(targeted therapy)
















Results











period











compound



03011)






07047




vol 1938(1)35 2018 DOI 101186s40880-018-

0299-7

(1)


Session 6 O 4




1



Background







68
















































Results











































69














Acknowledgement


02NO001219



101515bchm319943758497


1996 101016S0968-0004(06)80021-6


12 2006 101038nature05162


vol 31 19-24 2016 101016jcoph201608011


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Des vol 2 17-26 1997 101016S1359-0278

(97)00010-2


Session 6 O 5






1

Ryszard Rzepecki1




O-201621BNZ400541

Background








controlling it






















70






tive regions




















further experiments


and data analysis
















analysis




Results












Kc and S2





ndash Ser25






















transcription

71


DOI 101016jexger201009002


2838(18) 2018 DOI 101128MCB00181-18


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Session 6 O 6





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2 Ewa Maria Kratz

1








72






Session 6 O 7


of cosmetics



Background







































73




Results























nisms are necessary


Session 6 O 8






2

Andrzej Gamian1




Background




































74








Results













healthy controls























DOI 101111j1574-695X200600137x



0070539


17 1474-1483 1999 DOI 101016S0264-

410X(98)00385-5


77




2 Mirosław Anioł

2 Jolanta Saczko

1





Background




















anticancer activity

















Results

































78
























201621BNZ901904


235 710-717 2010 doi101258ebm2010009359


466-479 2007 doi101016jfoodchem2006

11054


2019 doi 103390molecules24040679







Wroclaw Poland

Background























injury















79







zymography

Results









































BNZ303151]


1727-1737 1992 10116101CIR8661727



S0828-282X(06)70315-4


77 544ndash555 1999 101007s001099900031




ajpheart007612002




12 Nathan Tancula

1 Agnieszka Orkusz

12 Remigiusz

Olędzki12




Background











80

























structure





























1072-1














Information Sheet











81




12 Nathan Tancula


12

Agnieszka Orkusz12




Background






























accessory

Results












sample


























petroleum ether

82










Chem 78 243-248 2001




548 2008 DOI 101007s11745-008-3181-6







18(1)5969 2019 DOI 102903jefsa20205969






Background































Results





















83





myelin sheath


12159-12165 2016 101021jacs6b04826



685617


101103PhysRevE79061502





1 Piotr Dobryszycki

1



Background























84













Limited proteolysis






minutes

Results


purification




respectively
















867oC

Limited proteolysis








incubation





















Acknowledgments









364-380 2015





85








1 Grzegorz Sawicki

2 Michael

Mosser3





Saskatchewan Canada

Background











ischemia
























86

















Results





























27BNZ400601


vol81-10 2019 doi 105527wjnv8i11





vol1021344-50 2018 doi 101097TP

0000000000002188


vol8 339-47 2012 doi 101038nrneph201283


vol1214210-21 201 doi 101172JCI45161




87





2




Background































3LBK) [3]

Results



compounds


binding

[kJmol]

Inhibition

constant

A - 36 765 nM

B - 35 821 nM

C - 31 4 M

D - 31 4 M

E - 31 3 M

F - 35 843 nM









Table)









tested before[1]

Acknowledgments


SUB008019029

88





Chem vol 60 4234-4244 2017



9 1104-1111 2010 104161cc9610956







1 Agnieszka Piwowar

1































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90


















genesis 2011 vol 32 no 11 pp1724-1733








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1 Aneta Starzec

2 Adam Junka

1



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Background









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Results






















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4(3) 58 2017 doi103390medicines4030058


40(4) 277-283 2015



doi103389fmicb201602161


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vol 10(1) 45 2017 doi103390ma10010045



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2 Hanna Baurska

2 Mirosław Anioł

3





Background















a phytoestrogen










Results
















92




References





changes

Kinga Chybicka12



3

Wojciech Kałas1






93


Results
















Acknowledgements





201269-97 2014 doi 101007978-3-642-54490-

3_18


doi 101186s12885-016-2925-6



for 3D cell culture



2




Background





















be variable [2 3]




















94




































Results






7DOX cells















vol 53(4) 294-295 2014




Industry vol 2(2) 68-75 2018



vol 4(2) 133-61 2015 DOI 103390

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Results














































96






Financial support







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200912014




124-133 2017 doi 101080216239452017

1320002





101186s13098-019-0494-y





2 Jarosław Całka

1




Background














domestic pigs




























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Results






























NNZ400348)




Active

neuronal

substance

Type of

plexus

Control

group

Indomethacin

group

PACAP

MP 969 plusmn

037

1191 plusmn 013

OSP 1051 plusmn

031

1402 plusmn 021

ISP 1282 plusmn

035

1636 plusmn 026



animals





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them with TNF-α

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Conclusions







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1




Gliwice Poland

99











processes


1 Ksenia A Sedova

2 Jan E Azarov

1 3


100

Background














rat hearts


















CVs

Results
















respectively)








conduction system








1 Brożyna Malwina

1 Junka Adam

1




Background







101

















activity [3]






















Kikgel)









Results

















ginosa










Conclusions















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NaOCl [45]




STMD23020053



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Advances vol 33(8) 1547-1571 2015 DOI



Zakażeń vol 10(1) 1-30 2019 DOI 1015374

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downregulation







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Results






specificity


































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Syndrome therapy



1 Ryszard

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Background







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nucleus






progerin
















gene signaling




















ndash lonafarnib











microscopy




Results






specificity









observed












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efficiency









1





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Background



























Results















disease

Acknowledgement





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carcinoma cells



1 Katarzyna


1 Julita Kulbacka

2 Joanna Rossowska

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mune cells












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1 Karol P Nartowski

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escitalopram





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disorder

Aim






signaling pathway






















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pathway








the infection






1









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12 Maciej Chyb


1

Magdalena Chmiela1






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1






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of curcumin
























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1 Adach K

2 Włoch A

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2 Adam Wojcieszonek

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2



Background






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in rats






1































palliative surgery






major complications



121
































References














2



Background


























Results




122











serum



3 Iwona Bil-Lula

4





Background




















observed




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Results












123





















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antibodies
























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before damage

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treatment



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was 26 microgm3





























12 Zuzanna Sycz

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life















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should be expanded


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1 Komorniak Natalia

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2 Styburski Daniel

2 Konecka Nina

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and organic farming








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3 Marta Skoacuterka

3

Daniel Styburski3








































128






















food nutrition









12(1) 82-89






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2 Laura Dallolio

2 Krystyna Pawlas

1



Bologna Italy

Background



















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children











meal



















Results








and scoring

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day - 1




















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relief


2 Tabassum Khan

2 Munira Momin

2 Soham


3 Sharmi Majumdar

3



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appropriately

Results












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effects of NSAIDs






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2653-2667 doi 101007s11095-016-1991-4





80 doi 101007978-3-030-15444-8_5




2 Wojciech Szlasa

1 Julita Kulbacka

2 Jolanta Saczko

2



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scratch assay

Results





133















1 Joanna Palma

1 Paula


1 Małgorzata Sarna

1 Robert Budawski

1 Wojciech


2 Daniel Styburski

2 Nina Konecka

3





Background






134






























milk proteins









repetitions

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threshold


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2 Branislav



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135





Kingdom

Background










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pressed tumor cells

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future

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vol 14 279-280 doi101038s41565-018-0356-z

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1 A Redzicka

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138










R1 = -C4H9 -C6H5 R2 = -Cl -Br


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2 Marcin Jędryka

34 Ewa Maria Kratz

1





Background











progression




















Results
















(p=0005 p=0016 p=0014 plt0001





(r=034 plt0001) CRP and IL-6 (r=058

















140









94 2019 DOI 101016jajog201902033





83 2018 DOI 101016jbpobgyn201804001





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practice




2


2 Joanna Palma

1 Nina Konecka

3





Background




























141








brain tumor surgery


2 Natalia Przybyła

1 Marta Kęsik

1 Natalia


2 Joanna Palma

2 Daniel Styburski

3 Marta Skoacuterka

3


142

Background

















after brain surgery














Polish Population

Results















surgery







2 Marcin Staszuk


2





Background

















143








of bacteria




















coatings

Results




































hospital infections






0500-00-Z09817


377 228-241 2011 DOI 101016S0140-

6736(10)61458-4



DOI 101016S0010-8545(02)00034-6






144





2 Jarosław Całka

1




Background








[1]








the porcine jejunum



























Results


























1994 doi 101016S0950-351X(05)80226-5

145







2












1 Paweł Turowski

1



1 Tomasz Bryniarski

1


1 Michał Wszoła

1


146

Background































staining)

Results










5206 respectively










below 80


32(8) 773-785 2014 doi101038nbt2958


1168-1177 2009 doi101002biot200900004


15(23) 2329-2336 2008 doi10217409298670

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Source of financing




2NCBR2017



and perspectives


2



147

































b Olga Wesołowska

b Krystyna Michalak

b


b





148





syndrome




Background























symptoms




[3]



PCOS
























Results



149












































1 Magdalena Żak

1






























literature





150












conditions











00159-2018








Background















[1 2]














Results

















(Figure)



151












Perkin Trans 2 (10) 1443-1447 1989

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1 Benita Wiatrak

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Total

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r = 009936

p = 06768

LDL-

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r = 0225

p = 03403

White blood

count

r = -03164

p = 01741

HDL-

cholesterol

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p = 04647

Platelet

count

r = 03544

p = 01252

Non-HDL-

cholesterol

r = 02143


r = -01964

p = 04066



















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10

20

30

40

50

370 390 410 430 450 470 490 510 530 550

Inte

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Milicz 80




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20

30

40

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Inte

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Groszowice 8


Milicz 80




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276

Index of authors

Adach K 116 218



Anioł M 77 91 197

Antczak O 171

Antoniak M A 60

Augoff K 97

Azarov J E 99

Baczyńska D 77 197

Bagaylyuk L B 258

Balevičiūtė A 59

Banaacuteš D 134


Banaś K 79 81 181 182 185 186 243

Barg E 166 177 202 270


Baurska H 91


Becan J 268

Belcarz A 45


Berman A 145

Bernikova O G 99

Bielak K 83

Bil-Lula I 52 78 85 122 151 180 208 254 262

Bizoń A 148



Błażkoacutew S 87

Bodnarchuk Yu V 258



Boszkiewcz K 88

Brand I 41

Brożyna M 90 100 187 225

Bryndal I 268

Bryniarski T 145

Brzozowska E 232

Budawsk R 133


Całka J 96 144 191 207

Chajduk E 66

Chmiel K 137

Chmiela M 110 111 112

Choromanska A 234

Chrząstek A 236


Chyb M 111 112

Chybicka K 92

Cierluk K 58 93 204

Cierzniak A 94


Cywoniuk P 145

Czajkowska M 96


Ćwilichowska N 194

Dabrowska A 38

Daczewska M 46 105

Dallolio L 128

Del Giudice A 43

Dembowski J 237

Dobryszycki P 83

Dobrzański T 145

Dobrzyńska D 186

Dobrzyński M 271

Dobrzyński W 271

Dorociak M 97

Dorosz M 66

Duda A 98 142

Durkina A V 99

Dutchak U M 212

Dydak K 90 100 187


Dzida K 226

Eiamphungporn W 248

Elias M 105

Emhemmed F 202

Fecka I 225

Fedorowicz S 271


Fernandez C 134


Filik K 232

Fornasier M 43

Franik G 148

Gabrielska J 226

Gajda M 35

Galantini L 43

Gamian A 73


Gebuza M 123

Geneja M 34 47 106

Gieysztor E 245

Gilowska I 71



277

Gliński P 208

Gliszczyńska A 205

Gładysz O 210

Gołkowska A 107

Gołyszny M 108

Gomoacutełka M 145


Gorczyca N 171


Goszczyński T 37


Gryziński M 66

Grzesik M 177

Grzeszczak A 115

Grzywacz K 116


Gula D 117

Gulla B 214

Guziński M 53

Gwiazdowska A 72

Halecka P 133

Harasym J 79 81 181 182 185 186 243


Heinrich A 119

Hejnowicz M 120

Hładyszowski J 220

Hof M 226

Hołub S 121


Ivantsiv O R 258

Iwankiewicz J 91


Jabłońśki S 174

Jacewicz D 38


Jana B 96 144 191 207

Janik M 122

Janiszewska E 71

Jankowska A M 93

Jarocki M 123

Jędryka M 139 219

Jędrzejak M 124

Junka A 90 100 187

Jurga A 194

Jurkowska K 237

Kabaj M 125 136 229 241

Kaczorowska A 92

Kalinowski L 38

Kaliszewski K 94

Kałas W 92

Kałduńska J 126 133 140 141 190 215

228 274

Kanclerz G 48

Kapczuk P 127 190 215 228 274

Karolewicz B 35 107

Kawalec A 128 130

Kazimierczak W 46

Kepinska M 33 40 117 134 159 272

Kęsik M 141

Khan N A 49 131

Khan T A 28 49 131

Kichler K 232

Kicia M 235

Kielan W 53

Kiełbasa E 117


Kikut J 126 133 140 141 190 215 228 274

Kizek R 26 134 223 248 252

Klak M 145

Klimas K 125 136 229 241

Kłosiński T 137

Kmieć Z 38

Kokot I 71 139

Komorek P 41

Komorniak N 118 126 127 133 140 141

190 215 228 263 274

Konecka N 126 133 140 141 190 215 228


Kordylewska I 144

Kosowska K 145

Kotas M 250

Kotowska A 155

Kotyra Ł 225

Kowalska P 145

Kowalski K 146

Kozakiewicz M 35

Kozioł A 147

Kozłowska J 77 197

Kral T 226

Krasoń M 148

Kraszkiewicz W 149

Kratz E M 71 139 219

Krawczyk M S 150

Krotkiewski H 219

Kroacutel M 33

Krupa A 110

Krześniak M 165



Kulbacka J 46 51 53 56 58 106 121

278

132 146 153 169 172 198 199 204 256

Kurzyna W 154

Kuzan A 155

Kuźma J 194

Kwiecień A 156

Lachowicz S 171

Lenda R 158

Leszek J 64



Lewoń D 161

Ligenza A 154

Lipke K 162

Lochyński S 72

Lubojański A 271

Ludyga T 108

Lupa D 163

Łabowska M B 93


Łukaszewicz M 174

Machowska M 102 103

Machynia M 166

Madej P 148

Magnus Rae J 167

Majerz I 150 156 238 239

Majgier K 169

Majumdar S 131

Malyško V 59



Małysa A 179

Markowski A 170

Maruszewska A 274

Matczyszyn K 62 82


Matoszka M 240

Matuszczyk I 165

Matuszewska A 119

Matuszewska K 166

Matuszewski M 237

Matyśkiewicz P 154

Mazur B 214

Mazurek W 120

Menezes S 49



Michalicha A 45

Michel O 58 132 172

Mielko K A 174




Milczanowska A 154

Milnerowicz H 33 40 134 159 161 183

Miskiv V A 258

Młynarz P 174

Molnar J 257

Momin M 49 131

Mooney J 233

Moreira H 166 177 202 270

Mosser M 85

Mucha I 260

Muller C D 202

Murauska A 178

Murgia S 43

Naporowski P 73


Nawaryński M 260

Nawrot K C 51


Niezgoda N 205

Novickij J 59

Novickij V 59 178

Nowak K 48

Nowak S 62

Nowakowska K 155

Nowicka J 271

Nowiczuk E 124

Nussberger S 38

Nyk M 51 60

Obuchowicz E 108

Ochnik M 64

Olbromski M 218

Olchowy A 179

Olchowy C 179

Olejnik A 52 78 151 180


Olędzki R 79 81 181 182 185 186 243

Ołdakowska M 183

Orkusz A 79 81 181 182 185 186 243

Ożyhar A 67

Pajączkowska M 271


Pakhomova O 29



Palma J 126 133 140 141 190 215 228 274

Palus K 191

Pałka M 63 69 192 247

Patel K 131

279


Pawlak A 92

Pawlas K 128 130

Pawlik K 62

Pecuch A 245

Peregrym K 272

Piasecki E 64


Piksa M 62

Pireddu R 43

Piscitelli G 128

Piszko P 194

Piwowar A 88 139 162 236 237

Ploska A 38

Płaczkowska S 122

Płochocki M 53

Pormańczuk K 208

Potyrak K 272

Pruchnik H 195 267

Przybyła N 141


Przychodzen P 38


Pyra A 268

Pytel J 201

Radajewska A 202


Raszewski Z 203

Redzicka A 137


Rogalski K 87

Rojek J 82

Rossowska J 34 37 47 106 153


Rusin M 165

Rutkowska M 119



Rybak Z 271

Rychlicka M 205

Rząp D 207

Rzepecki R 63 69 102 103 192 247

Saczko J 58 77 132 172 204


Sarna M 133

Sarzyński D 210

Sawicka E 88 154 225 231 237

Sawicki G 85

Schilleacuten K 43

Sedova K A 99

Sehnal K 134

Seweryn P 179

Shah S 131

Shchur M B 211

Siadak A 195

Sinico C 43

Skibiński P 210

Skora J S 211 212

Skorupska A 67

Skorupski M 214



215 228 274

Smardz J 179

Smoleński M 216

Sobczyński M 64

Sobierajska P 271

Sochocka M 64


Sołkiewicz K 219

Sonetha V 131

Spiegel M 220 222

Spinek A 250

Sroka A 156

Sroka Z 220 222

Staigvila G 178


Starzec A 90 225

Starzyńska A 225

Stasiak S 225

Staszuk M 142

Stebnicka A 155


Strub D J 72

Strugała P 226

Styburski D 126 127 133 140 141 190

215 228 274

Supplit S 58

Surowiak A K 72

Sycz Z 125 136 229 235 241

Szafraniec M 231

Szczepańska P 231

Szczukowski Ł 272



Szewczyk A 46 56 58 204

Szewczyk M 133

Szlasa W 58 132 172 204

Szota M 233

Szwedowicz U 234

280

Szydełko M 185


Szyjka A 177


Szymańska M 239

Szymański S 238

Szymonowicz M 271






Świtalska M 37

Tancula N 79 81 181 182 185 186 243

Tarek M 25 58 172 204

Targońska S 271

Telenga M 245

Tokarek B 246


Tomczak A 63 69 192 247


Trukan A 225

Tunikowska J 27

Turek K 123

Turowski P 145

Tyliszczak M 250

Tymicki G 145

Tyszer J 250



Uryga A 189

Vasyliuk V M 212

Vlasiuk T I 212


Wadowska K 254

Waglewska E 255

Wala K 256

Wanarska E 42


Weżgowiec J 179

Węgierek K 34 37 47 106

Wiatr M 258

Wiatrak B 177 272

Więckiewicz M 179

Wiglusz J 271

Wiglusz K 260 271

Wiglusz R J 260

Wiśniewska K 262

Witkowska D 73 231

Włoch A 195 267

WłochA 116

Włodarczyk M 112


Wojnicz D 229

Wolańska E 245

Wolska A 175 265 274

Worobiec M 267

Woźniak M 122

Woacutejcicka A 268

Woacutejcik M 69

Woacutejciuk G 66

Woacutejciuk K 66

Wrona A 149


Wrzyszcz A 262

Wszoła M 145

Wysoczańska A 270

Zając P 272


Zajkowicz A 165

Zakrzewski W 271

Zalewski J 48


Zaręba N 272

Zborowska A 272

Zdrojewska M 274

Želvys A 59

Zhurakivska O V 212



Zieliński M 108


Zioło E 92

Złocińska A 35

Zmijewski M A 38

Zoglowek A 83

Zwolińska K 64

Zygmunt A 115

Żak A 62

Żak M 149

Żbikowska B 220

Żwierełło W 126 127 133 140 190 215

228 274

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